analytical approaches for mcpd esters and glycidyl esters...

Food Additives & Contaminants: Part A2012, 1–35, iFirst

Analytical approaches for MCPD esters and glycidyl esters in food and biological

samples: a review and future perspectives

C. Crewsa, A. Chiodinib*, M. Granvoglc, C. Hamletd, K. Hrncirıke, J. Kuhlmannf, A. Lampeng, G. Scholzh,R. Weisshaari, T. Wenzlj, P.R. Jastib and W. Seefelderh

aThe Food and Environment Research Agency (FERA), Sand Hutton, York YO41 1LZ, UK; bILSI Europe a.i.s.b.l., Avenue E.Mounier 83, Box 6, B-1200 Brussels, Belgium; cGerman Research Institute for Food Chemistry (DFA), Lise-Meitner-Straße 34,D-85354 Freising, Germany; dPremier Foods, Lord Rank Centre, Lincoln Road, High Wycombe HP12 3QR, UK; eUnilever,Olivier van Noortlaan 120, NL-3130 AC Vlaardingen, the Netherlands; fSGS Germany GmbH, Weidenbaumsweg 137D,D-21035 Hamburg, Germany; gFederal Institute for Risk Assessment, Thielallee 88–92, D-14195 Berlin, Germany;hNestle Research Centre, Vers-Chez-les-Blanc PO Box 44, CH-1000 Lausanne, Switzerland; iChemisches undVeterinaruntersuchungsamt, Schaflandstraße 3/2 und 3/3, D-70736 Fellbach, Germany; jInstitute for Reference Materials andMeasurements, European Commission, Directorate-General Joint Research Centre, Retieseweg 111, B-2440 Geel, Belgium

(Received 12 April 2012; final version received 8 August 2012)

Esters of 2 - and 3-monochloropropane-1,2-diol (MCPD) and glycidol esters are important contaminantsof processed edible oils used as foods or food ingredients. This review describes the occurrence and analysis ofMCPD esters and glycidol esters in vegetable oils and some other foods. The focus is on the analytical methodsbased on both direct and indirect methods. Methods of analysis applied to oils and lipid extracts of foods havebeen based on transesterification to free MCPD and determination by gas chromatography-mass spectrometry(indirect methods) and by high-performance liquid chromatography-mass spectrometry (direct methods). Theevolution and performance of the different methods is described and their advantages and disadvantages arediscussed. The application of direct and indirect methods to the analysis of foods and to research studies isdescribed. The metabolism and fate of MCPD esters and glycidol esters in biological systems and the methodsused to study these in body tissues studies are described. A clear understanding of the chemistry of the methods isimportant when choosing those suitable for the desired application, and will contribute to the mitigation of thesecontaminants.

Keywords: chloropropanols; MCPD; MCPD esters; glycidol; glycidyl esters; food processing; carcinogens;vegetable oil; deodorisation; LC-MS; food safety; chromatography

Introduction

This review offers a detailed perspective of develop-

ments in analytical methods for fatty acid esters

of chlorinated propanols and glycidol. Chlorinated

propanols, especially 3-monochloropropane-1,2-diol

(3-MCPD), have been known as food contaminants

for over 30 years. Glycidol has long been recognised as

a carcinogen, but has only relatively recently been

shown to be associated with 3-MCPD in foods. Serious

attention has turned to the esters of MCPD and

glycidol in the past few years following their discovery

in refined vegetable oils.A review of MCPD and glycidyl esters is important

on account of the high levels found in relation to their

free forms, making it important to find out more about

their occurrence and possible toxicity, to monitor

mitigation successes, and eventually to ensure the

future compliance with any exposure recommenda-tions. Analytical methods have advanced considerablyin recent years, thus there is considerable benefit inproviding an update of the current status and identi-fying knowledge gaps. This review was produced bymembers of the International Life Sciences InstituteTask Force on Process Related Compounds/NaturalToxins Task Force and Risk Assessment of Chemicalsas part of a joint activity on 3-MCPD esters infood products and is published in association with apaper describing the potential mitigation measures(Craft et al. Forthcoming 2012).

History of MCPD esters and glycidyl esters

A number of chlorinated propanols were discoveredas contaminants of acid-hydrolysed vegetable protein

*Corresponding author. Email: [email protected]

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(acid-HVP), used as a flavour enhancer, by Velıseket al. (1979) and Davıdek et al. (1980). Acid-HVP ismanufactured by the hydrochloric acid hydrolysis ofvegetable material (typically the residue from oilseedpressing) carried out under conditions of high temper-ature. The chlorinated products are formed fromthe reaction of the acid with residual lipids. Themajor compound, 3-MCPD, was suspected to be acarcinogen, and its discovery led to the developmentand validation of analytical methods, followed by foodsurveys and toxicological testing. Recommendationsregarding acceptable levels of intake were issued byofficial bodies including the European CommissionScientific Committee on Food (European Commission2001a, 2001b) and the US Food and DrugAdministration (USFDA) (2008). Industry action hasled to a reduction of 3-MCPD in commercial foodproducts such that they now comply with regulationsand contribute to a reduction in exposure.

Esters of 3-MCPD (also referred as ‘‘boundMCPD’’) were also found in acid-HVP and in modelsystems when precursors such as triacylglycerols(TAG) were reacted with hydrochloric acid (Velıseket al. 1979; Davıdek et al. 1980, 1982). They were alsoreported in goat’s milk by Cerbulis et al. (1984), but nofurther effort was made to investigate their occurrenceuntil quite recently, when significant amounts of3-MCPD fatty acid esters were found in foods and inparticular in refined edible oils.

Structures of MCPD esters

MCPDs are chlorinated analogues of glycerol havinga chlorine atom in positions 1 or 2. MCPD esters cantake many forms, being either mono- or di-esters, asshown in Figure 1, of the range of fatty acids found inoils, and they can be expected to mimic the naturalTAG in the composition of their fatty acids. Thechloropropanol portion also exists as racemic mix-tures, and as both 2 - and 3-chloro propyl esters.

Dichloropropanol esters have not been reported inrefined oils and it is unlikely that they are formedduring current manufacturing processes.

Toxicity of MCPD esters

The toxicological significance of 3-MCPD esters andtheir contribution to dietary intake of 3-MCPD is notknown (Bakhiya et al. 2009). Dietary intake and fateare of concern because of the potential contributionto the dietary intake of 3-MCPD from the hydrolysisof 3-MCPD esters in vivo by lipases in the gastrointes-tinal tract. There has not yet been a toxicologicalstudy of 2-MCPD as pure standards have not beenreadily available until recently and because in termsof occurrence the free 2-MCPD is usually less abun-dant. It exceeds 3-MCPD only in acid-HVP that hasundergone hydrolysis to reduce 3-MCPD, a process towhich 2-MCPD is more resistant.

Glycidyl esters

Fatty acid esters of glycidol (3-hydroxy-1,2-epoxypro-pane) (Figure 2) are of concern as glycidol is known tobe carcinogenic in animal studies and is thought to actvia a non-threshold, genotoxic mechanism of action(Bakhiya et al. 2009; Habermeyer et al. 2011). Glycidolhas been classified as probably carcinogenic to humans(Group 2A) by the International Agency for Researchon Cancer (IARC) (2000) and is ‘‘reasonably antici-pated to be a human carcinogen’’ by the US NationalToxicology Program (NTP) (1990, 2007).

The current interest in glycidyl esters arose frominvestigations into the discrepancies between theresults for different methods for the determination of3-MCPD esters observed when the same sampleswere analysed by two methods. It was proposed andconfirmed that overestimation of the levels of 3-MCPDesters was the result of the chlorination by addedsodium chloride of additional compounds identifiedas fatty acid esters of glycidol (Kuhlmann 2008;Weisshaar and Perz 2010).

The Bundesinstitut fur Risikobewertung (BfR –German Federal Institute for Risk Assessment) hasconcluded that infants who are fed exclusively on

Cl

HO

HO

OH

Cl

HO

(a) (b)

Cl

HO

R1O

Cl

R1O

R2O

R2O

Cl

R1O

(c) (d) (e)

Figure 1. Structures of fatty acid esters of 3-MCPD:(a) 3-MCPD; (b) 2-MCPD; (c) 3-MCPD mono-ester;(d) 3-MCPD di-ester; (e) 2-MCPD mono-ester. R1,R2¼ acyl of fatty acid.

Figure 2. Structure of a fatty acid ester of glycidol(R¼ alk(en)yl).

2 C. Crews et al.

commercially prepared infant milk formula could takein harmful levels of glycidol (BfR 2009).

Determination of MCPD esters and glycidyl esters

The separation of fat components, especially acylgly-cerols, has classically been carried out using thin-layerchromatography (TLC), and this technique was thefirst used to separate chloropropanol esters frommodel systems and food lipids (Velısek et al. 1979;Davıdek et al. 1980). The chloropropanol ester frac-tions were extracted from the TLC plates with solventand determined by gas chromatography-mass spectro-metry (GC-MS).

TLC methods combined with silica column chro-matography were used by Gardner et al. (1983) toshow the presence of 3-MCPD esters in adulteratedcooking oils in Spain. The esters were determineddirectly by GC-MS. A similar TLC/silica columnchromatography clean-up technique was applied totarget 3-MCPD esters in goat’s milk by Cerbulis et al.(1984). In this case, acid-catalysed transesterificationwas used to produce fatty acid methyl esters and free3-MCPD, which were both determined by GC-MS.

Hydrolysis methods were used more widely in 2004.Divinova et al. (2004) used acid-catalysed transester-ification (methanolysis) to release free 3-MCPD fromfat extracts of food, and phenylboronic acid (PBA)derivatisation to measure the total and the free3-MCPD (in the whole portion of food) by GC-MS,from which the bound level was calculated by differ-ence. Hamlet and Sadd (2004) used enzymatic cleavageto release free 3-MCPD from bound forms in fatextracts of cereal products. GC-MS with heptafluor-obutyrylimidazole (HFBI) derivatisation was used tomeasure 3-MCPD in both the whole sample and thehydrolysed fat portion.

Following concerns regarding the possible forma-tion of 3-MCPD during acid-catalysed transesterifica-tion, a method based on transesterification underalkaline conditions using sodium methoxide wereproposed (Weisshaar 2008). This method was sub-jected to further study and modification to preventdegradation of 3-MCPD under the alkaline conditionsand to deal with the issue of formation of 3-MCPDfrom the sodium chloride added as a part of theprocedure. Alkaline-catalysed transesterification hasbeen adopted as an official method by the DeutscheGesellschaft fur Fettwissenschaft (DGF – GermanSociety for Fat Research), however acid-based proce-dures are still widely accepted.

Separation of 2- and 3-MCPD mono-esters fromdi-esters is of considerable interest but has so far beenapplied only on a limited scale using silica columnfractionation (Zelinkova et al. 2007). Analysis ofmono- and di-ester fractions can clearly provide more

information of use in toxicological studies, as can thedetermination of individual esters.

Esters of glycidol can be determined indirectly byits conversion to free MCPD. Subtraction of the freeMCPD derived fromMCPD esters from the sum of thefree MCPD released by transesterification of theMCPD esters and the glycidyl esters gives the level ofthe glycidyl ester content. A non-stoichiometric factorreflecting the transformation of glycidol to 3-MCPDmust be applied (DGF 2011a).

Indirect methods can give only a measure of thetotal MCPD ester content of a food sample, or thesum of MCPD ester and glycidyl esters. It might beassumed that direct methods, where there is notransesterification stage, should be both easier andprovide more detailed information, but in practice themethodology has proven challenging to apply andthe large number of potential MCPD esters meansdata analysis can be difficult.

Direct methods were initially based on GC-MSanalysis of fractions isolated by TLC and/or columnchromatography (Reece 2005; Zelinkova et al. 2007),but the use of liquid chromatography-mass spectro-metry (LC-MS) has proved more popular and conve-nient. Procedures have ranged from direct injectionof oil solutions to the incorporation of solid-phaseextraction clean-up. Glycidyl esters and MCPD esterscan be determined simultaneously (Haines et al. 2011).For the independent determination of glycidyl esters,LC-MS is the method of choice, typically with gelpermeation chromatography clean-up (Dubois et al.2011; Granvogl and Schieberle 2011b).

MCPD esters are assumed to be associated with thenaturally occurring acylglycerols in foods, and solventextraction of the non-polar oil fraction of foods is thefirst step in most analytical methods, although somepolar element is preferred to aid extraction of themono-esters. In the early years quantification washampered by a lack of standards, but these are nowavailable from a small number of suppliers.

The direct determination of glycidyl esters is easierthan that of MCPD esters on account of the singleposition for fatty acid esterification and the fact thatthe esters can be separated by gas chromatography(GC) or liquid chromatography-mass spectrometry(LC-MS) after isolation of a non-polar lipid fractionand separation from the acylglycerols fraction. Indirectmethods are linked to the determination of MCPDesters and based on conversion of glycidyl esters to free3-MCPD which is determined in the usual ways.

Analytical methods for monitoring

Indirect methods

The principle of indirect determination of MCPDesters is the conversion of a number of individual

Food Additives & Contaminants: Part A 3

MCPD esters of fatty acids into a single compound,3- or 2-MCPD, that is quantified. To date, severalindirect methods have been developed for the analysisof MCPD esters, the vast majority as 3-MCPD esters.

The analytical protocol of these methods comprisesa uniform series of steps: addition of an internalstandard (either free or esterified form of isotopicallylabelled 3-MCPD) to the sample, transesterification(commonly performed either in acid or alkalinemedium), neutralisation of the reaction mixture andsalting out (using different neutralising reagents andsalts), derivatisation of the cleaved 3-MCPD/2-MCPD,and GC-MS analysis.

Analytical protocol for indirect methods

Since even small variations within each individualanalytical step may have a significant impact on thespecificity, repeatability, reproducibility, truenessand other parameters of the method, the analyticalprotocol is treated in detail in this section.

MCPD cleavage

The indirect determination of bound MCPD in oils orlipid extracts commences with the cleavage of MCPDesters. Free MCPD can be released enzymatically(Hamlet and Sadd 2004), but the majority of themethods employ a chemical route using a transester-ification step. Transesterification in the presence ofmethanol (methanolysis) results in the conversion ofTAGs and partial acylglycerols into fatty acid methylesters (FAMEs) and glycerol. Simultaneously, 3- and2-MCPD esters are converted to free 3- and 2-MCPD,respectively. The transesterification step can be carriedout both in acid (Divinova et al. 2004) and alkaline(Weisshaar 2008) media.

The alkaline-catalysed transesterification approachis convenient owing to its short duration (with reactiontimes up to 10min). The major drawback of thisapproach is that 3-MCPD is unstable in alkalinesolutions giving rise to glycidol. Two independentstudies have shown degradation of 3-MCPD to berapid under these conditions; the 3-MCPD recoveryfound after 1min of transesterification was 83–95%(Hrncirık et al. 2011), 75% after 3min (Kuhlmann2011a), about 50% after 9min (Kuhlmann 2011a), and40–41% after 10min (Hrncirık et al. 2011). It can beexpected that the poor stability of 3-MCPD in alkalinemedia affects the sensitivity of these methods signifi-cantly, thus there may be scope to optimise theprotocol to achieve maximum recovery by minimisingtransesterification time. However, assuming that thedecomposition of 3-MCPD (released from its nativeesters) and its deuterated analogue (used as internalstandard) occurs at the same rate, the transesterifica-tion time should not have an impact on the trueness

of the method. Nevertheless, the levels of bound3-MCPD found at very short transesterification times(1–2min) were surprisingly 10–20% higher than thoseobtained at longer times (5–10min) (Hrncirık et al.2011). The authors attributed this phenomenonto different conversion rates amongst the various3-MCPD forms (e.g. mono-esters being converted to3-MCPD faster than di-esters), and, as a consequence,suggested choosing a deuterated di-ester form of 3-MCPD as internal standard because of the similaritywith the majority of the native 3-MCPD esters.

In order to eliminate the problems related to unde-sirable conversion of MCPD to glycidol, Kuhlmann(2011a) proposed performing the alkaline-catalysedtransesterification at a temperature below �22�C,which resulted in a higher recovery of 3-MCPD atthe expense of a prolonged transesterification time(18 h). In contrast to alkaline-catalysed transesterifica-tion at ambient temperature, no degradation of3-MCPD was observed during acid-catalysed transes-terification (Hrncirık et al. 2011).

Neutralisation and salting out

Salting out, a step that follows transesterification,facilitates the extraction of lipophilic compounds(FAMEs) from the transesterified mixture. The mostcommon salting out agents used in the analysis of3-MCPD are sodium chloride, sulphate salts andsodium bromide (Divinova et al. 2004; Weisshaar2008; Hrncirık et al. 2011; Kuhlmann 2011a). Theseare added to the reaction mixture either after or duringthe neutralisation.

This step is particularly critical for the specificity ofmethods based on alkaline transesterification. In 2008,the specificity of a newly developed method based onalkaline catalysis and salting out by sodium chloride(Weisshaar 2008) was questioned by demonstratingthat the content of 3-MCPD esters was overestimatedin some samples due to the additional 3-MCPD formedde novo during sample preparation (Kuhlmann 2008).The compound leading to overestimated levels wasconfirmed to be glycidol, which can be present inrefined oils as esters of fatty acids (Weisshaar andPerz 2010).

This finding became the basis for an attempt todevelop a new methodology for the indirect estimationof the level of glycidyl esters in oils. Its principle wasthe calculation of the difference between two indepen-dent determinations: the first one (A) leading to thequantification of the sum of 3-MCPD and glycidylesters (after their conversion to 3-MCPD), while thesecond one (B) was specific to 3-MCPD esters. Suchan approach provides meaningful results only if theformer determination (A) ensures a complete conver-sion of glycidol into 3-MCPD, whilst in the specificmethod (B) any such glycidol conversion is avoided.

4 C. Crews et al.

While the latter can be achieved relatively easy eitherby replacing sodium chloride by other salts duringsalting out (e.g. DGF 2011a; Kuhlmann 2011a) or bythe deliberate decomposition of glycidyl esters priorto transesterification (DGF 2009), the achievementof a complete conversion of glycidol is more difficultbecause it is dependent on both the type of salts andthe conditions applied during the salting out step.

This holds particularly true for pH. Hrncirık et al.(2011) demonstrated that by modifying the pH duringsalting out, the glycidol conversion could vary greatly –it was limited in neutral solutions, but considerablyhigher at pH 2. By monitoring the pH of ‘‘neutralised’’mixtures derived from the application of differentmethods, it can be noticed that the pH varies substan-tially. Therefore, although these methods are based onthe assumption of a complete conversion of glycidol,it is very probable that this will not be always the caseand that the results will depend on the method used.

Unlike the methods employing alkaline transester-ification, the methods based on acid-catalysed transes-terification are not affected by variations in theconditions during salting out (Hrncirık et al. 2011).Glycidyl esters are irreversibly degraded during theacid-catalysed transesterification and do not interferewith the determination of MCPD esters (Hrncirık et al.2011).

Derivatisation and analysis

Due to the low volatility (b.p. 213�C) and high polarityof 3-MCPD, derivatisation prior to the GC analysisis preferable as it greatly improves sensitivity by theformation of a volatile 3-MCPD derivative andspecificity by enabling monitoring of specific fragmentions of the derivative. Several straightforward methodshave been proposed and used since the methodologyfor the analysis of free 3-MCPD was developed in theearly 1990s. The most common derivatisation agentused is PBA, followed by HFBI. Both derivatisationagents represent good alternatives, although HFBI issensitive to moisture and the procedure must be carriedout under anhydrous conditions. It should be notedthat both derivatisation methods enable simultaneousdetermination of both 3 - and 2-MCPD. Alternativemethods used to derivatise free 3-MCPD, such as theformation of dioxolane compounds with acetone, havenot yet been applied to MCPD ester determination.

The most common methods used

Different combinations of the analytical stepsdescribed above, and also additional modificationsto each individual step led to the development of anumber of methods. Table 1 provides an overviewand a basic description of the most common indirectmethods that have been published to date.

Methods based on acid-catalysed transesterification

Methods based on acid-catalysed transesterificationare derived from the pioneering work of Divinova et al.(2004), who applied methanolysis by a mixture ofsulphuric acid and methanol. Since then several mod-ifications have been adopted (Zelinkova et al. 2006;Seefelder et al. 2008; Hrncirık et al. 2011). A certaindisadvantage of this analytical approach is the rela-tively long time of transesterification (16 h), meaningthat it is typically carried out overnight. All acid-transesterification based methods show good robust-ness, i.e. the variation of analytical conditions has alow impact. Comparable results have been obtainedregardless of the changes in salting out (Ermacora andHrncirık 2012a) or the derivatisation procedure(Seefelder et al. 2008). More recently the same authorsincluded an additional step (conversion of glycidylesters to 3-monobromobropanediol (3-MBPD) estersinto their protocol) that enabled glycidyl esters to bequantified simultaneously with 3- and 2-MCPD esters(Ermacora and Hrncirık 2012b).

These methods are specific to 3- and 2-MCPDesters. Recently, the interference of chloride ions andglycidyl esters was evaluated under the conditions ofthe acid catalysed method (Hrncirık et al. 2011). Thestudy demonstrated that 3-MCPD esters were over-estimated only when unrealistically high levels ofchloride ions were added to the samples. Glycidylesters did not interfere with the determination of3-MCPD esters, which corroborates claims of the highspecificity of these methods. Very recently, Miyazakiet al. (2012) successfully employed a lipase fromCandida rugosa for the hydrolysis of 3-MCPD estersand glycidyl esters. Glycidol was subsequently con-verted to 3-MBPD, and following the derivatisationby PBA it was quantified next to 3-MCPD by thestandard GC-MS analysis.

Method based on enzymatic hydrolysis

An alternative to acid-catalysed transesterification isthe enzymatic cleavage of 3-MCPD esters. Hamletand Sadd (2004) applied lipase-catalysed hydrolysis of3-MCPD esters using a lipase from Aspergillus oryzae(24 h incubation at neutral pH). Coupled with deriva-tisation by HFBI and subsequent GC-MS analysis, themethod was used for the determination of MCPDesters in cereal products. Despite a good sensitivity andrepeatability, no further applications of enzymes forthe analysis of MCPD esters have been reported.

Non-specific methods based on alkaline-catalysedtransesterification

The transesterification by sodium methoxide (durationup to 10min) introduced by Weisshaar (2008) led toconsiderable shortening of the sample preparation


Table

1.IndirectanalyticalmethodsforMCPD

andglycidylesters.

Methoddenomination

Analyte

Internalstandard

Transesterification

(tim

e)Derivatisation

agent

Comments

References

Acidic

3-M

CPD

esters

PP-3-M

CPD-d5

Sulphuricacid/m

ethanol

(16h)

PBA

Divinovaet

al.(2004),

Zelinkovaet

al.(2006)

Acidic

3-M

CPD

esters

P-3-M

CPD-d5

Sulphuricacid/m

ethanol

(16h)

PBA/H

FBI

Seefelder

etal.(2008)

Acidic

3-and2-M

CPD

esters,

glycidylesters

PP-3-M

CPD-d5,

glycidyl-P-d5

Sulphuricacid/m

ethanol

(16h)

PBA

Hrncirıket

al.(2011),

Erm

acora

andHrncirık

(2012a,2012b)

Enzymatic(A

spergillus

oryzae)

3-and2-M

CPD

esters

5-�-C

holestane

Lipase

(24h)

HFBI

Applied

tobakerygoods

HamletandSadd(2004)

Enzymatic(C

andida

rugosa)

3-M

CPD

andglycidyl

esters

3-M

CPD-d5,

3-M

BPD-d5

Lipase

(0.5h)

PBA

Miyazakiet

al.(2012)

Alkaline,

non-specific

(DGF

C-III

18

(09)A)

Sum

of3-M

CPD

and

glycidylesters

3-M

CPD-d5

Methoxide/methanol

(5–10min)

PBA

DGF(2009)

Alkaline,

non-specific

(DGF

C-V

I17(10))

Sum

of3-M

CPD

and

glycidylesters

3-M

CPD-d5

Methoxide/methanol

(5–10min)

PBA

DGF(2011a)

Alkaline,

withpre-treat-

ment(D

GF

C-III

18

(09)B)

3-M

CPD

esters

3-M

CPD-d5

Methoxide/methanol

(5–10min)

PBA

Method

withdrawn

in2011

DGF(2009)

Alkaline,

chloride-free

(DGF

C-V

I18(10))

3-M

CPD

esters

PP-3-M

CPD-d5

Methoxide/methanol

(3–5min)

PBA

DGF(2011b)

Alkaline,

mild(SGS)

3-and2-M

CPD

esters,

glycidylesters

PP-3-M

CPD-d5,

3-M

BPD-d5

NaOH/m

ethanol(18h)

PBA

Kuhlm

ann(2011a)

6 C. Crews et al.

time. The method, combined with salting out withsodium chloride, derivatisation with PBA and analysisby GC-MS was adopted as the method DGF C-III18 (09) A (DGF 2009). Following the validation bycollaborative trial (Fiebig 2011), the method wasmodified and adopted as DGF C-VI 17 (10) (DGF2011b). The major drawback of this method is that itis not specific to bound 3-MCPD, as it determinesthe sum of 3-MCPD esters and glycidyl esters.

Specific methods based on alkaline-catalysedtransesterification upon acid pre-treatment

In order to overcome the drawback of the lackof specificity mentioned above, an additional pre-treatment step by sulphuric acid/propanol mixturewas introduced to selectively eliminate glycidyl esters.After the acid pre-treatment, the protocol followed themethod DGF C-III 18 (09) A (DGF 2009) describedin the section on ‘‘Occurrence of glycidyl esters.’’ Themethod was introduced as DGF C-III 18 (09) B, but itwas withdrawn early in 2011 as the pre-treatment stepwas proven to be insufficient for reliable determinationof 3-MCPD esters (Fiebig 2011; Shimizu et al. 2011).

Specific methods based on alkaline-transesterificationand chloride-free salting out

Another way to tackle the issue of specificity is tocombine alkaline-based transesterification with saltingout, in which a salt other than sodium chloride is used.This approach has been applied, with various modifi-cations, by several laboratories and finally adoptedas a DGF C-VI 18 (10) method (DGF 2011a). Thesemethods are specific to 3-MCPD, but the degradationof 3-MCPD during the transesterification step (dis-cussed in section ‘‘MCPD cleavage’’ in detail) remainsa point of concern.

Specific methods based on mild alkaline-transesterification

Performing the alkaline transesterification at lowtemperature virtually eliminates the undesirable con-version of MCPD to glycidol (Kuhlmann 2011a),although the transesterification time is prolonged to18 h (and is thus comparable with acid-catalysedtransesterification). In addition to the determinationof both MCPD isomers, the method enables thesimultaneous determination of the total glycidyl estercontent. This seems to be a promising approach, butthe complex analytical protocol and strict applicationof subfreezing conditions required in the first part ofthe sample preparation increase the chance of errors.So far the method has been performed only by a singlelaboratory, and its performance requires evaluationby collaborative trial.

Methods for indirect determination of glycidyl esters

The choice of a method for the indirect determinationof glycidyl esters is relatively limited. One option is adifferential approach that employs the application oftwo independent analyses: one that determines the sumof 3-MCPD esters and glycidyl esters using sodiumchloride for salting out (DGF 2011b) and a specific onethat determines only 3-MCPD esters. From the differ-ence of both determinations, a total amount of glycidylesters can be calculated; however, due to the reasonsdiscussed in section on ‘‘Direct methods,’’ the accuracyof such an approach still requires to be verified.Another, more elaborated alternative is the use of abromide salt that enables the conversion of glycidol to3-MBPD, as applied in the mild alkaline method,enzymic method and the method based on acid-catalysed transesterification (Table 1).

Kusters et al. (2011) developed a method for thesimultaneous determination of 3-MCPD esters andglycidyl esters in foods based on conversion ofthe glycidol moiety into 3-methoxypropane-1,2-diol(3-MPD) by acidic methanolysis prior to rapid alka-line-catalysed transesterification at ambient tempera-ture and incorporating an isotope-labelled 3-MCPDester as internal standard. The method was validatedfor various fat-rich foodstuffs.

Proficiency tests

In 2009 the Joint Research Centre (JRC) of theEuropean Commission conducted a proficiency teston 3-MCPD ester analysis in oils (Karasek et al. 2010).The test provided the first snapshot that reflected thesituation regarding the analysis of 3-MCPD esters atthat time. Participants of the test, which included,amongst others, commercial, industrial and officialcontrol laboratories, were not restricted in terms of themethod used. Two samples were analysed in the test:a virgin olive oil spiked with 3-MCPD dioleate (bound3-MCPD level 4.58mg kg�1) and a fully refined palmoil (assigned value of bound 3-MCPD: 8.77mgkg�1).In total, 29 out of 34 participants (85%) providedresults which were considered satisfactory (absolutez-score 52) for the spiked sample. The outcome of theanalysis of refined palm oil was much less favourablein that only 56% of participants complied with theperformance criteria. Further analysis of the dataindicated an obvious impact of the analytical methodon the outcome. All results obtained by acid-transesterification based methods were satisfactory(five datasets, in the range of 6.7–9.1mgkg�1).Specific alkaline-transesterification based methods,either those using acid pre-treatment or thosechloride-free, provided much larger variation (3.0–16.5mg kg�1). However, 11 out of 15 datasets werefound compliant. Nine laboratories which appliedthe non-specific alkaline-catalysed transesterification


based method failed, presumably due to the interfer-ence of glycidyl esters present in the refined oil sample.

Another more recent proficiency test was organisedby Food Analysis Performance Assessment Scheme(FAPAS) (2011). Twenty-six datasets were obtainedfor the analysis of 3-MCPD esters in a refined palm oilsample (assigned value¼ 4.7mg kg�1). Compared withthe previous proficiency test, the results showed onlylimited improvement of the situation: 16 datasets(62%) complied with the minimum performance crite-ria (absolute z-score 52) with reported values in therange of 3.5–6.1mg kg�1, 10 laboratories failed,in seven cases due to the same reason as in the caseof the JRC proficiency test, i.e. the use of non-specificalkaline-transesterification based methods.

Gaps and limitations

There has been enormous progress in the developmentof indirect methods for the determination of MCPDesters in the last decade, and in particular in last fouryears. Advances in the methodology have led to theimprovement of the performance (e.g. sensitivity) ofmethods used and to a better understanding of thelimitations of these methods. Substantial effort on thedevelopment of methods based on different principles,and their further modifications, has resulted in a largenumber of different methods being currently available.The fact that these methods differ in their scope andperformance is a cause of some concern. It would behighly desirable to harmonise current analytical meth-odology for MCPD and glycidyl esters and to identifythose methods that meet certain performance criteria.Such harmonisation will not only improve the qualityof the results, but also simplify their communication.To achieve this objective, it seems inevitable thatselected methods will be validated within internationalcollaborative studies and ultimately adopted as officialmethods.

Direct methods

The first data reported on the 3-MCPD ester contentof edible oils were generated by transesterification,isolation and derivatisation of the free 3-MCPDmoiety followed by GC-MS determination. However,it was soon apparent that the data generated werenot comparable and that results depended much onthe procedure applied to specific food matrix types(Karasek et al. 2010). For this reason it would beadvantageous to develop analysis methods that donot require chemical reactions and that allow thedirect measurement of the esters. Well-establishedsample preparation procedures, which are traditionallyemployed in lipid analysis, could be applied dueto the similarities between 3-MCPD esters andacylglycerides.

A challenge for the direct determination of MCPDesters is the large number of substances that have to beincluded into the assays. The distribution pattern ofthe 3-MCPD esters and glycidyl esters is expectedto follow the profile of the fatty esters of the productinvestigated. This complexity of the analyte composi-tion requires selective separation and detection meth-ods, and compared with the indirect methods, largerinvestment in reference materials.

Description of the methods

In the following paragraphs, analysis methods for thedirect determination of glycidyl esters and/or 3-MCPDesters are compiled and discussed. Informationwas extracted from the scientific literature and frompresentations given at recent conferences on this topic.An overview of the methods is given in Table 2.It should be emphasised that method performancecharacteristics presented, such as limit of detection(LOD) and limit of quantification (LOQ), are based onthe respective esters, be they glycidyl esters or 3-MCPDesters and not the free compounds.

The analyte pattern of glycidyl esters is rathersimple as they contain only one fatty acid moiety.Dubois (2011) concluded from theoretical consider-ations that the glycidyl fatty acid ester content in mostedible oils can be sufficiently well characterised by theanalysis of seven glycidyl fatty acid ester species. Theseare the glycidyl esters of lauric, myristic, palmitic,stearic, oleic, linoleic and linolenic acids.

Masukawa et al. (2010) described the determina-tion of five different glycidyl esters (in particularpalmitic, stearic, oleic, linoleic and linolenic acid ester)in edible oils by liquid chromatography atmosphericpressure chemical ionisation mass spectrometry(LC-APCI-MS) in positive-ion mode. The authorsalso considered the application of tandem massspectrometry, but abandoned this approach due tothe lack of selective fragment ions. Sample preparationof the oil samples was based on extraction of theanalytes with acetonitrile followed by dual stagesolid-phase extraction (SPE), employing firstly a C18and secondly a silica cartridge. The authors reportedthat the application of the SPE cartridges in this orderprovided superior results compared with the applica-tion in reverse order. A sub-2mm particle size C18column and an ultra-high-performance liquid chroma-tography system (UHPLC) were employed for theseparation of the glycidyl esters.

The direct injection of a diluted TAG-rich oilsample into the LC-MS system led to fast deteriorationof system performance. The recoveries of glycidylesters spiked into an edible oil sample were dependenton the species and were in the range from about 75%to about 95%. The instrument detection limit (IDL)was estimated for each of the five glycidyl esters to be

8 C. Crews et al.

Table

2.DirectanalyticalmethodsforMCPD

andglycidylesters.

Methoddenomination

Analyte(s)

Calibration

Sample

preparation

Chromatographic

column

Mobilephase

composition

Comments

References

LC-M

S,SIM

,APCI

inpositive-ionmode

Glycidylesters

Internalstandardisation

withglycidyl-P-d31

Dilutionof250mgoilin

5ml

acetone

ODS-A

MC18,3mm

ID�150mm,3mm

particle

size

(YMC-Pack)

SolventA:MeO

H/A

CN/

H2O¼4.25/4.25/1.5;

solventB:acetone

Collisonand

Blumhorst(2011),

Blumhorstet

al.

(2011)

LC-TOFMS

Seven

glycidyl

esters

Standard

addition

Dilutionofsample

incyclo-

hexane/ethylacetate,GPC

clean-uponBio-BeadsS-

X3,GEcontainingfrac-

tionevaporatedand

reconstitutedin

acetone;

additionalclean-upforoils

withhighDAG

andMAG

content:SPEon500mg

silica

(eluentCH

2Cl 2)

AcquityHSST3,

2.1mm

ID�50mm,

1.8mm

particle

size

(Waters)

SolventA:

MeO

H:H

2O¼3:1;

solventB:iso-PrO

H,

both

containingform

icacid

ComparisonofLC-M

S/

MSwithLC-

HRTOFMS;glycidyl

laureate

andglycidyl

myristate

included

Duboiset

al.(2011)

LC-TOFMS

Seven

glycidyl

esters;17

3-M

CPD

mono-and

di-esters

Standard

addition

Dualsample

preparation:(a)

glycidylesterand3-

MCPD

monoester:dual

stageSPE:firstly2gC18

(eluentACN),then

500mg

silica

(eluentCH

2Cl 2);(b)

3-M

CPD

diesters:column

chromatographyon3g

silica

gel

(eluentCH

2Cl 2)

AcquityHSST3,

2.1mm

ID�50mm,

1.8mm

particle

size

(Waters)

SolventA:

MeO

H:H

2O¼3:1;

solventB:iso-PrO

H(both

containingammo-

nium

form

ate

and

form

icacid)

Differentsample

prepa-

rationforMCPD

mono-anddi-esters

Dubois(2011)

LC-M

S,SIM

,APCI

inpositive-ionmode

Seven

glycidyl

esters

Isotopedilutionwith

deuterated(2)and

13C-labelled(5)ana-

logues

ofeach

analyte

Dilutionofsample

inn-pen-

tane:diethylether¼95:5,

clean-upbyfractionation

onsilica

gel

column,

evaporationofsolvent,

andreconstitutionofresi-

duein

acetonitrile

2mm

ID�150mm,

3mm

particle

size

(Phenomenex)

SolventA:0.1%

form

icacidin

H2O;solvent

B:0.1%

form

icacidin

ACN

Highsolventconsump-

tion,poorrecoveries

forsomeanalytes,

butcompensation

withlabelledstan-

dardsforeach

indi-

vidualstandard

Granvogland

Schieberle(2011a)

LC-TOFMS:ESI,SIM

ofsodium

ion

adducts*,positive-

ionmode

Fiveglycidyl

esters,tw

enty

3-M

CPD

ester

mono-and

di-esters


withOO-M

CPD-d5

andglycidyl-P-d31

Dilutionofsample

inauto-

samplervialwithHPLC

solventB

containing

internalstandards

LunaC18column,

3mm

ID�50mm,

3mm

particle

size

(Phenomenex)

SolventA:MeO

H:0.26mM

NaAcin

MeO

H:A

CN¼8:1:1;

solventB:

CH

2Cl 2:0.26mM

NaAc

inMeO

H:A

CN¼8:1:1

‘‘Dilute-and-shoot’’

approach;problems

withinstrumentsta-

bility,corrosionof

ionsourceparts

Haines

etal.(2011)

LC-TOFMSESI,SIM

ofsodium

ion

adducts*,positive-

ionmode

Fiveglycidyl

esters,nine

3-M

CPD

ester

mono-and

di-esters


with1-L-3-M

CPD-

d5,PP-3-M

CPD-d5

andglycidyl-O-d31

Liquid/liquid

partitioningof

edible

oilin

n-hexaneand

acetonitrile,SPEofn-

hexanephase

onSep-Pak

PlusSI,500mg(W

aters),

eluentsCHCl 3,andSPEof

acetonitrile

phase

onSep-

PakPlusC18,500mg

(Waters),eluents

acetoni-

trileandethylacetate,

mergingofeluents,evapo-

rationofsolventand

reconstitutionofresiduein

acetonitrile

AcquityUPLC

BEH

C18,2.1mm

ID�50mm,1.7mm

particle

size

SolventA:0.2mM

l�1

sodium

form

ate

inH

2O:M

eOH¼85:15;

solventB:0.2

mM

l�1

sodium

form

ate

inMeO

H:H

2O¼97.5:2.5

Horiet

al.(2012)

(continued

)


Table

2.Continued.

Methoddenomination

Analyte(s)

Calibration

Sample

preparation

Chromatographic

column

Mobilephase

composition

Comments

References

LC-M

S/M

S,SRM,

APCIin

positive-ion

mode

Seven

glycidyl

esters,tw

enty

3-M

CPD

ester

mono-anddi-

esters


Dilutionofsample

inTBME:diethylether¼4:1,

clean-uponsilica

SPE

cartridge,

eluentn-hex-

ane:diethylether¼94:6

Shim

adzu

UFLC,

ODS4.6mm

ID�150mm,

5mm

particle

size

SolventA:10mM

ammo-

nium

form

ate

and0.1%

form

icacidin

MeO

H;

solventB:iso-PrO

H

MacM

ahonet

al.

(2011)

LC-M

S,SIM

,APCIin

positive-ionmode

Fiveglycidyl

esters

Externalcalibration;

standardsin

MeO

H/

iso-PrO

H(1/1)

Double

SPE:firstcolumn:

Sep-PakVacRC

C18,

500mg(W

aters),second

column:Sep-PakVacRC

Silica,500mg(W

aters)

evaporationofeluentand


MeO

H/iso-PrO

H(1/1)

ODS,4.6mm

ID�150mm,

5mm

particle

size

SolventA:ACN/M

eOH/

H2O¼17/17/6;solvent

B:iso-PrO

H

Methodsuitableforfats

andoils;lower

recoveriescompared

withMasukawa

etal.(2010)

Masukawaet

al.

(2011)

LC-M

S,SIM

,APCIin

positive-ionmode

Glycidylesters


standardsin

MeO

H/

iso-PrO

H(1/1)

Double

SPE:firstcolumn:

Sep-PakVacRC

C18,

500mg(W

aters),second

column:Sep-PakVacRC

Silica,500mg(W

aters),



MeO

H/iso-PrO

H(1/1)

AcquityUPLC

BEH

C18,2.1mm

ID�100mm,1.7mm

particle

size

(Waters)

SolventA:ACN/M

eOH/

H2O¼17/17/6;solvent

B:iso-PrO

H

Fast

deteriorationof

system

perform

ance

withTAG-richoil

samples

Masukawaet

al.

(2010)

LC-O

rbitrapMS,ESI,

SIM

ofammonium

ionadducts,positive-

ionmode

Twelve3-M

CPD

mono-and

di-esters


withPP-3-M

CPD-d5

and1-P-3-M

CPD-d5

Dualsample

preparation:(a)

3-M

CPD

monoester:SPE

on500mgaminopropyl

cartridge(eluentn-

hexane:CH

2Cl 2:diethyl

ether¼89:10:1);(b)3-

MCPD

di-esters:column

chromatographyon1.8g

silica

gel

(eluentn-hex-

ane:ethylacetate¼85:5)

Kinetex

minibore

C8,

2.1mm

ID�50mm,

1.7mm

particle

size

(Phenomenex)

SolventA

10mM

ammo-

nium

form

ate

inwater;

solventB

10mM

ammonium

form

ate

inMeO

H

Low

detectionand

quantificationlimits

for3-M

CPD

di-

esters,poorionisa-

tionefficiency

for

3-M

CPD

monoesters

Moravcovaet

al.

(2012)

10 C. Crews et al.

DART-O

rbitrapMS,

SIM

ofammonium

ionadducts,positive-

ionmode

Twelve3-M

CPD

mono-and

di-esters


withPP-3-M

CPD-d5

and1-P-3-M

CPD-d5

Dualsample

preparation:

(a)3-M

CPD

monoester:

SPEon500mgaminopro-

pylcartridge(eluent

n-hexane:CH

2Cl 2:

diethylether¼89:10:1);

(b)3-M

CPD

di-esters:

columnchromatography

on1.8gsilica

gel

(eluent

n-hexane:ethyl

acetate¼85:5)

––

Higher

LODsand

LOQscompared

withUHPLC

method,veryshort

analysistime

Moravcovaet

al.

(2012)

LC-M

S/M

S,SRM,ESI

inpositive-ionmode

Fourteen

3-M

CPD

mono-and

di-esters

Standard

addition

Dilutionofsample

inCH

2Cl 2:M

eOH¼1:1

LunaC18column,

3mm

ID�50mm,

3mm

particle

size

(Phenomenex)

SolventA:3mM

NH

4Ac

inMeO

H:A

CN¼9:1;

solventB:

CH

2Cl 2:A

CN:3mM

NaAcin

MeO

H:A

CN¼8:1:1

Severematrix

effects

Pinkstonand

Stoffolano(2011)

LC-M

S,SIM

,APCIin

positive-ionmode

Fiveglycidyl

esters


andinternalstan-

dardisationwith

C17:0-G

E,standards

inMeO

H/iso-PrO

H(1/1)

Double

SPE:firstcolumn:

Sep-PakVacRC

C18,

500mg(W

aters),second

column:Sep-PakVacRC

Silica,500mg(W

aters),



MeO

H/iso-PrO

H(1/1)

ODS,4.6mm

ID�150mm,5mm

particle

size

SolventA:MeO

H;solvent

B:iso-PrO

HFurther

developmentof

methodpresentedby

Masukawaet

al.

(2011);interferences

experienced;

improved

LODs

Shiro,Kondo,

Kibune,

etal.

(2011)

LC-M

S,SIM

,APCIin

positive-ionmode

Fiveglycidyl

esters


standardsin

MeO

H/

iso-PrO

H(1/1)

Double

SPE:firstcolumn:

Sep-PakVacRC

C18,

500mg(W

aters),second

column:Sep-PakVacRC

Silica,500mg(W

aters),



MeO

H/iso-PrO

H(1/1)

ODS,4.6mm

ID�150mm,5mm

particle

size

SolventA:

MeO

H:H

2O¼92:8;sol-

ventB:iso-PrO

H

Shiro,Kondo,

Masukawa(2011)


below 20 pg on-column. However, a drawback of themethod was the application of external calibration foranalyte quantification, which is not able to compensatefor matrix effects encountered.

In the following paper, Masukawa et al. (2011)proposed a slightly modified analysis procedure for thedetermination of the five aforementioned glycidylester species. The main changes were the dilution ofthe sample in a mixture of acetone and chloroformthat also enabled the analysis of solid fats, and theapplication of conventional high-performance liquidchromatography (HPLC) instead of UHPLC. Thelatter modification aimed to broaden the group ofpotential users, as it does not require specialisedinstrumentation. However, the authors noted thatapplying the initial clean-up procedure with thechange of UHPLC to HPLC resulted in lower analyterecoveries. They attributed this to interferences stem-ming from residual TAGs. The detection capability ofthis method was improved compared with the originalmethod by a factor of three, which might be theconsequence of the higher injection volume applied inthe HPLC procedure. The recovery values of glycidylesters spiked into an edible oil were slightly above100% for the modified procedure. Repeatabilityrelative standard deviations for all analytes had amaximum of 4.0%.

The same group of authors proposed a furthermodification of the method, which resulted in short-ened analysis times and again improved detectioncapability (Shiro, Kondo, Kibune, et al. 2011). TheIDLs of this third method were about eight times lowerthan the IDLs of the original method. The LOD of theanalytes in edible oil were estimated to range between25 and 33 mg kg�1. Another improvement was theelimination of chlorinated solvents. The acetone/chlo-roform mixture was replaced by tert-butyl methylether/ethyl acetate (4:1 v/v), and the eluent of thenormal phase SPE clean-up was n-hexane/ethyl acetate(95:5 v/v). The application of glycidyl heptadecanoateas internal standard did not improve analytical preci-sion compared with external calibration. In a presen-tation given at the 102nd AOCS Annual Meeting (1–4May 2011 in Cincinnati, OH, USA) the authorsdescribed interferences experienced with the thirdanalysis procedure (Shiro, Kondo, Masukawa 2011).In particular glycidyl linoleate eluted with almost thesame retention time as an unknown substance, whichseemed to be extracted from plastic parts of the SPEmanifold. A slight modification of the HPLC mobilephase composition remediated this problem withoutaffecting method performance for the other analytes.

UHPLC with high resolution time of flight massspectrometry (UHPLC-TOFMS) analysis with internalstandardisation with one isotope-labelled glycidyl esterand two isotope-labelled 3-MCPD esters was per-formed by Hori et al. (2012) for the simultaneous

determination of five glycidyl esters, three 3-MCPDmono-esters and six 3-MCPD di-esters in edible oils.Their sample preparation encompassed liquid–liquidpartitioning of the edible oil in n-hexane and acetoni-trile, and subsequent solid-phase extraction on silicaand C18 cartridges. In contrast to the methoddescribed above, the two cartridges were used inparallel. The n-hexane phase was loaded onto thesilica cartridge, whereas the acetonitrile phase wasapplied to the C18 cartridge. The collected eluents werecombined, evaporated and reconstituted in acetonitrileprior to the measurement. Interferences as describedby Shiro, Kondo, Masukawa (2011) were not reported,which could be attributed to the higher mass spectro-metric resolution of the applied TOF instrument.

None of the analysis procedures mentionedabove has been validated for the direct determinationof glycidyl laurate and glycidyl myristate, which areexpected to occur in some oils such as palm kernel oilor coconut oil. This gap was recently filled by Duboiset al. (2011). The method comprised gel permeationchromatography (GPC) of the diluted edible oilsample, which provides sufficient separation of glycidylesters from the bulk matrix. GPC clean-up wascomplemented by SPE in silica for samples containingpalm kernel oil or coconut oil. The authors consideredthe SPE clean-up necessary in order to separate theglycidyl ester fraction from co-eluted short chaintriacyl- and diacylglycerols (DAG), as it improvedanalytical precision and stability of the instrument. Themeasurements of the sample extracts were performedby both LC-TOFMS and liquid chromatography-tandem mass spectrometry (LC-MS/MS).

The latter technique did not show advantagescompared with LC-TOFMS as collision induced dis-sociation (CID) provided only non-selective fragments.Quantification was performed by external quantifica-tion as well as by standard addition, and internalstandardisation by five isotope-labelled glycidyl esters.The authors identified standard addition as the mostsuitable quantification method. This was becausematrix effects could be compensated for in the absenceof isotope-labelled analogues which were not yetcommercially available. Recoveries and precisionof the analyses were assessed on spiked safflower oilsamples. Recoveries of all analytes were above 70%in almost all experiments, whereas intermediate preci-sion was usually better than 25%. Recoveries for fiveof the seven glycidyl esters were quite stable overthe concentration range (0.1–7.5mg kg�1) tested. Onlyglycidyl laurate and glycidyl myristate had highervariability.

These two glycidyl fatty acid esters are also part ofthe scope of an analysis procedure developed at theDeutsche Forschungsanstalt fur LebensmittelchemieLeibniz Institut (DFA – German Research Centre forFood Chemistry). Granvogl and Schieberle (2011a)

12 C. Crews et al.

reported at the 102nd AOCS Annual Meeting amethod that enables the determination of sevenglycidyl ester species by stable isotope dilution analysisLC-MS. Sample clean-up consisted of fractionation onsilica gel 60 (16 g) containing 7% of water and stepwiseelution of the analytes with pentane/diethyl ether (95/5v/v). The fraction between 150 and 250ml wascollected, evaporated to dryness and reconstituted inacetonitrile. A pentafluoropentylpropyl stationaryphase and a water/acetonitrile mobile phase gradientwere employed for the HPLC separation of the glycidylesters. Analytes were detected by positive-ion APCI-MS in selected ion monitoring mode (SIM).

Recovery of the analytes was rather poor. Thelowest recovery value was for glycidyl linoleate,at about 34%. The advantage of the application ofisotope dilution is that analyte losses during samplepreparation are corrected for. On the other hand thestandards cannot influence LOD and LOQ. Details onmethod performance characteristics other than recov-ery were not provided. A similar procedure applyingsilica SPE cartridges instead of silica gel columnsfor sample clean-up was developed at the USFDA(MacMahon et al. 2011). They applied a standard C18column and methanol containing ammonium formateand formic acid for chromatographic separation,and APCI-MS/MS for the detection of the analytes.Method performance was evaluated on two differentdays, two spiking levels and for three different matri-ces. The recoveries reported ranged between 79% and114%, whereas the relative standard deviation of theanalyses was between 3% and 16%.

Haines et al. (2011) and Collison and Blumhorst(2011) applied the least degree of sample preparationfor the determination of glycidyl esters in vegetableoils. It consisted only of the dilution of the edible oilin an organic solvent prior to the injection into the LCsystem. The method also enabled the determinationof a range of 3-MCPD esters, which will be describedbelow. The determination of the analytes wasperformed by electrospray ionisation LC-TOFMS inSIM. Analytes were separated on a C18 stationaryphase applying a mobile phase gradient consisting ofmethanol, acetonitrile, and methylene chloride.Sodium acetate was added to the mobile phase inorder to generate sodium ion adducts of the analytes.The limits of detection of glycidyl esters in palm oilwere about 100 mg kg�1, with the exception of glycidylmyristate for which the LOD was about 290 mg kg�1.Hence, the LODs were about three times higher thanthose reported by Shiro, Kondo, Kibune, et al. (2011).The precision of the analysis procedure, expressed asrelative standard deviation, was reported to lie between5% and 10%. Unfortunately, no information wasprovided on the recovery of the analytes from differentoils, which could vary significantly due to matrixeffects.

A major drawback of the dilute-and-shoot

approach chosen by Haines et al. (2011) is the use of

low levels of sodium salts in the mobile phase to drive

the formation of the sodium adduct ion. The high load

of matrix results in the need for frequent instrument

cleaning and maintenance. Additionally, mobile phase

components have caused corrosion of some ion source

parts such as the ESI nebuliser needle. In a recent

update of the analysis procedure, methylene chloride

was replaced by non-chlorinated solvents, the sample

was diluted in acetone prior to injection, and LC-

APCI-MS was applied instead of LC-ESI-TOFMS

(Blumhorst et al. 2011; Collison and Blumhorst 2011).

The LOD varied for the seven glycidyl fatty acid esters

between 40 mg kg�1 of oil and 160 mg kg�1 of oil.Recovery values were determined for two edible oils

at three different fortification levels. They indicated

significant matrix effects, with a tendency to over-

estimation of the content of some analytes.The determination of 3-MCPD esters alone, or in

combination with glycidyl esters, provides additional

challenges compared with the determination of glycidyl

fatty acid esters alone. The challenges result not only

from the additional number of analytes that has to be

covered, but also by the strong similarity of the

analytes to major matrix constituents, in particular

monoacylglycerols (MAGs) and DAGs.Reports in literature on the direct determination of

MCPD esters are very limited. This might be attribut-

able to the lack of suitable commercial reference

materials in the early days of this topic. Laboratories

active in this field were until recently obliged to

synthesise reference materials themselves. Details of

the synthesis of glycidyl esters and/or MCPD esters

can be taken from several publications (Masukawa

et al. 2010; Granvogl and Schieberle 2011a; Haines

et al. 2011). However, the commercial supply of

reference materials has recently improved significantly.

A broad range of both native and stable isotope-

labelled glycidyl and MCPD esters is available

from different suppliers. A non-exhaustive list of

suppliers comprises (in alphabetical order) Chiron AS(Trondheim, Norway), Toronto Research Chemicals

(Toronto, ON, Canada), and Wako Pure Chemical

Industries, Ltd (Osaka, Japan).The Lebensmittelchemisches Institut des

Bundesverbandes der Deutschen Sußwarenindustrie

(LCI) (2008) published an equation for the calculation

of the number of possible 3-MCPD esters depending

on the number of different fatty acids contained in a

particular edible oil (LCI 2008). Hence, six different

fatty acids would theoretically provide 96 different

3-MCPD mono- and di-ester combinations, including

all possible stereoisomers. It is not realistic to expect to

be able to determine routinely all possible 3-MCPDesters. Hence, a compromise and agreement has to be


found as to which analytes should be included in theanalysis.


The papers presented so far have to be seen in viewof the lack of such agreement and the limitationsprovided by the limited availability of reference mate-rials. Haines et al. (2011) included five 3-MCPD mono-esters and fifteen 3-MCPD di-esters in their analysisprocedure, whereas MacMahon et al. (2011) presentedchromatograms of twenty-two 3-MCPD esters,Moravcova et al. (2012) analysed three 3-MCPDmono-esters and nine 3-MCPD di-esters, andPinkston and Stoffolano (2011) focused on in total14 mono- and di-esters of 3-MCPD, which wereaccording to them the most relevant. Hori et al.(2012) developed an analysis method for three3-MCPD mono-esters and six 3-MCPD di-esters.Dubois (2011) concluded from his considerationsthat the determination of ten 3-MCPD di-esterswould be sufficient to cover between 90% and 99%of the expected total 3-MCPD di-ester content,depending of the type of edible oil. Exceptions areprovided by coconut oil and palm kernel oil, whichcontain high amounts of short chain fatty acids.Additionally, seven 3-MCPD mono-esters should beincluded in the assay.

The analytical approaches applied for the determi-nation of 3-MCPD esters in edible oils differ widely.Haines et al. (2011) applied the dilute-and-shootapproach described above. They used deuteratedglycidyl palmitate as internal standard for 3-MCPDmono-esters and deuterated 3-MCPD-dioleate for3-MCPD di-esters. The LODs were between 15 and60 mg kg�1 for MCPD mono-esters, between 4 and10 mg kg�1 for 3-MCPD di-esters containing atleast one unsaturated fatty acid, and 25 mg kg�1 for3-MCPD di-esters containing only saturated fattyacids. The precision of the method, expressed as relativestandard deviation, was in the range of 5–10%.However, questions again arose concerning the stabil-ity of the analysis system and the matrix effectsencountered.

Pinkston and Stoffolano (2011) applied an analyt-ical procedure similar to that of Haines et al. Theirmodifications consisted of an ammonium salt mobilephase additive in place of the sodium salt used byHaines et al. (2011) and the application of tandemquadruple mass spectrometry operated in selectedreaction monitoring mode instead of LC-TOFMS.Use of the ammonium salt mobile phase additiveavoided the degradation of instrument performance,and associated cleaning and maintenance, described byHaines et al. (2011). Remarkably, they experiencedsevere matrix effects in the analysis of vegetable oilsamples, which caused them to choose quantification

by standard addition. The LOQs achieved were inthe best case about 20–50 mg kg�1 for mono-estersand 50–100 mg kg�1 for di-esters. However, LOQsof di-esters rose for some oil matrices to 500–1000 mg kg�1. For this reason, they recommend thatsample clean-up be included in future methods.

The disadvantages of using sodium salts in themobile phase were also tackled by Moravcova et al.(2012) who investigated alternatives. The addition ofammonium acetate to the mobile phase and conse-quently the measurement of ammonium ion adductswas identified as best solution and provided for3-MCPD di-esters detection limits comparable withthose of sodium ion adducts. The lower limits of theworking range of their UHPLC-OrbitrapMS methodwas for the 3-MCPD di-esters studied in the rangeof 2–5mg kg�1. Mass resolution was set to 50.000FWHM. The total run time of the UHPLC methodwas 8min. This was further reduced by applyingDART-OrbitrapMS for measurement of the 3-MCPDesters. Cycle times per sample were shortened to about20 s. Sample preparation for 3-MCPD di-ester analysisconsisted of column chromatography over silica geland was identical for both measurement techniques.However the sensitivity of the DART-OrbitrapMSanalysis was lower than that of UHPLC-OrbitrapMS.3-MCPD mono-esters were isolated from the matrixaccording to the procedure described by Seefelder et al.(2008). Neither the UHPLC nor the DART basedmeasurement method was sufficient sensitive for thedetermination of 3-MCPD mono-esters in real lifesamples.

A more complex analysis procedure was presentedby Dubois (2011). It consisted of the separate deter-mination of MCPD mono-esters and MCPD di-esters.Double SPE was applied for the isolation of MCPDmono-ester, whereas MCPD di-esters were separatedfrom the bulk by column chromatography on silica gel.Both MCPD ester fractions were measured by LC-TOFMS. Dubois confirmed the need to apply eitherisotope dilution with stable isotope-labelled glycidyland MCPD esters, or standard addition for compen-sating for matrix effects (Dubois 2011; Dubois et al.2011). Isotope dilution would be easier and faster thanstandard addition, but is hampered by the limitedavailability of labelled analogues of the analytes.An effect that cannot be compensated for by eithermethod is caused by differences of responses ofco-eluting isomers, which could cause significant bias.Dubois reported a difference of response between1-palmitoyl-3-MCPD and 2-palmitoyl-3-MCPD of40% (Dubois 2011). In addition the two estersco-eluted with palmitoyl-2-MCPD.

A more recent study (Dubois et al. 2012) comparedthe performance of an indirect and a direct method.The indirect procedure used acid-catalysed trans-methylation and HFBI derivatisation. The direct

14 C. Crews et al.

method had two separate extraction procedures, oneused double SPE cartridges to isolate 2 - and 3-MCPDmono-esters and glycidyl esters and the second useda single silica column to isolate 2- and 3-MCPDdi-esters. The esters were measured by LC-TOFMSand quantified using matrix-matched standard addi-tions. There was no significant bias when the methodswere used to analyse numerous samples of palm oils.The indirect method was considered more suitablefor routine application. Consequently, the trueness ofanalysis results has to be questioned. Unfortunately,the analytical community lacks a supply of edible oilwith certified glycidyl ester and MCPD ester content.For this reason, analysts have compared their resultswith those gained by other methods, including methodsbased on the indirect determination of glycidyl andMCPD esters. So far it has not been made clear whichmethod operated under which conditions providesresults closest to the true value.

Collison and Blumhorst evaluated the results of thedirect determination of glycidyl esters (Blumhorst et al.2011; Collison and Blumhorst 2011) in five edible oilsagainst the results obtained with a slightly modifieddouble SPE procedure proposed by Shiro, Kondo,Kibune, et al. (2011). The results gained by the twomethods gave similar results. However, their associatedmeasurement uncertainties values did not agree.

Discussion

It is not clear which method gives the ‘‘better’’ results.Questions regarding over- and underestimation of theanalyte contents by one or the other method have tobe tackled and answered in order to progress towardsa generally accepted analysis procedure.

The development of analytical methods for thedetermination of individual glycidyl esters in edibleoils is more advanced than the development of directanalysis methods for the determination of MCPDesters in oil. This might be explained by the simplertask and the easier access to reference materials, whichwere scarcely available in the early days of this topic.

Comparison of direct and indirect analytical methods

Introduction and background

Whereas initial analytical methods for MCPD andglycidyl esters were mainly based on the detection of3-MCPD and glycidol that could be released fromthe esters (indirect analysis), more recent advances inMCPD and glycidyl ester analysis have targeted thedirect determination of the individual compounds.This trend may be explained by the fact that indirectanalytical methods do not provide any detailed infor-mation on the chemical structure of the differentesters. This type of information may, however, become

relevant when the chemical structure of the estersmight turn out to have an impact on their biologicalfate or toxic potential, and, therefore, differentiationwould become necessary. The major triggers of thedevelopment of direct methods however are the con-cerns expressed on the precision and trueness ofcommonly applied indirect analytical methods forMCPD esters, whose performances are thought to becompromised by the analytical steps as mentionedabove. Even though promising, more accurate directanalytical methods have a limited applicability in aroutine environment mainly due to the relativelyhuge number of analytical standards they require.Comparison of direct and indirect analytical methodsmay therefore serve as an alternative for evaluating thetrueness of indirect analytical methods since certifiedreference materials are not yet available.

The following paragraphs provide an overviewon studies dealing with the comparison of direct andindirect analytical methods for MCPD and glycidylesters and gives furthermore recommendations whichmay be taken into consideration for promoting fitfor purpose and commonly recognised methods forMCPD and glycidyl esters.

Overview on comparative studies

Data on the comparison of direct and indirect analyt-ical methods have mainly been presented in the frameof conferences. Table 3 provides an overview on theoutcome of current studies.

Direct and indirect methods for glycidyl esters

The first comparison of direct and indirect analyticalapproaches for glycidyl esters was carried out byShimizu et al. (2011). The authors concluded thatthe results obtained from direct and indirect measure-ment of the glycidyl esters were not comparable.Commercially available samples in particular showedlower glycidyl ester levels or even negative results whenapplying the indirect method. For direct determina-tion, glycidyl esters were isolated by double SPEand measured by liquid chromatography coupled to asingle quadrupole mass spectrometer (LC-MS)equipped with an APCI interface. Calibration of thedirect analytical method was carried out externally(no internal/labelled standards). For measuring sam-ples indirectly, the DGF differentiating method in itsformer version C-III 18 (09) (DGF 2009) was applied,including the use of sodium chloride in the salting outand derivatisation step. The authors suggested that thepresence of sodium chloride in this procedure mayhave led to the formation of additional amounts of3-MCPD through the means of partial acylglycerides(MAG and DAG) or through residues of glycidylesters which were incompletely destroyed during the


Table

3.Comparisonofdirectandindirectanalyticalmethods.

Methodsdenomination

Analytescompared

Oilmatrices

included

Indirect

method

withdrawn,

yes/no

2-and3-M

CPD

differentiatedby

directmethod,

yes/no

Comments

References

DirectLC-M

S–double

SPE

versusIndirectDGF

C-III

18

(09)A;B

GE

4cookingoil,2palm

,2spread

Y–

Non-comparable

andinconclusive

results

Shim

izuet

al.(2011)

DirectLC-M

S-TOF;‘‘dilute

and

shoot’’versusindirectDGF

C-V

I18(10)A;B

GEandMCPD

esters

4palm

,1blend

NN

Significanthigher

resultsofMEs

bytheindirectmethod.

Comparable

resultsforthe

absence

ofglycidylester

Hinrichsen(2011)

DirectLC-M

S;double

SPEversus

directLC-M

S;‘‘dilute

and

shoot’’

GE

1palm

,1soybean,

1corn

––

Incanola

oiltheresultsmatched

nicely,in

palm

oilnot.A

special

shapeofthespraysourceis

recommended

for2.3.3-D

Collisonand

Blumhorst(2011)

DirectLC-M

S;‘‘dilute

andshoot’’

versusindirectDGF

C-III

18

(09)part

A

Sum

ofGEandMCPD

esters

2palm

,2blend

NN

Non-comparable

and

inconclusiveresults

Haines

etal.(2011)

DirectLC-M

S/LC-M

S2,SPE

versusindirectDGF

C-III

18

(09)A;Bversusindirect

GC-M

S,alkalinemild(SGS)

GE

2avocado,1sunflower,

1palm

,2DAG

rich,

1TAG

rich

2.3.1-I:Y;

2.3.3-I:N

–DirectGEsdeterminationin

oil

samplesshowed

bettercorrela-

tionwithSGS‘‘3-in-1’’than

withDGF

C-III

18(09)

Granvogland

Schieberle(2011a)

DirectLC-M

S2;‘‘dilute

and

shoot’’versusindirect

MCPD

esters

9vegetable

NN

Comparable

resultsforseven

of

nineinvestigatedvegetable

oil

samples.Indirectapproach

rather

isrelatedto

theDGF

C-V

I18(10)methodthanto

aWeisshaarmethod

Pinkstonand

Stoffolano(2011)

A)DirectLC-M

S2;GPC

andSPE

versusB)directLC-M

S/LC-

MS2,SPE(D

FA)versusC)

indirectGC-M

S,acidic

versus

D)indirectGC-M

S,alkaline

mild(SGS)versusE)indirect

DGF

C-V

I18(10)A;B

GEandMCPD

esters

A),B),C),D)10palm

;D),E)10soybean,

13sunflower,53

palm

N–

A),B),D)comparable

resultsfor

GEsfrom

somedirectmethods

versusindirect‘‘SGSindirect

3-in-1’’method.C),D),E)

comparable

resultsforallthree

indirectmethodsregarding3-

MCPD.Comparable

resultsfor

2-M

CPD

determined

indirectly

bytw

omethods(2-M

CPD

was

notevaluatedwithin

methodE)

Kuhlm

ann(2011b),

Granvogland

Schieberle

(2011b)

DirectLC-TOF;double

SPE

and

SGC

formono-M

Eanddi-ME

versusindirectGC-M

S,acidic

MCPD

esters

30palm

N(Y

)indirectly

Analysisofmono-estersin

30palm

oilsamplesbydirectandindi-

rect

methodsgavesimilar

results

Dubois(2011)

16 C. Crews et al.

acidic pre-treatment. Differences in the results from

both methods may therefore be largely explained by

the drawbacks of the withdrawn C-III 18 (09) method.

Also, the absence of internal standards for the direct

analysis of glycidyl esters prevented compensation for

losses of the analytes during sample preparation.A method based on GPC separation combined with

an additional clean-up by solid-phase extraction on

silica-gel and LC-MS detection has been validated

including four 13C-labelled (glycidyl palmitate, oleate,

linoleate and linolenate) standards for the analysis of

seven glycidyl esters in various edible oils by (Dubois

et al. 2011). The method has been compared with the

indirect analytical method for glycidyl esters known as

‘‘SGS 3-in-1 method’’ (Kuhlmann 2011a), which is

based on alkaline catalysed release of glycidol, fol-

lowed by a transformation of glycidol to MBPD,

derivatisation with PBA and analysis by GC-MS.

Results obtained from the analysis of 10 samples of

refined palm oil showed a good correlation of results,

however, accompanied by a systematic bias

(Kuhlmann 2011b).The same samples were further analysed by a stable

isotope dilution analysis based direct analytical

method developed at the DFA (Granvogl and

Schieberle 2011a) using seven 2H- or 13C-labelled

standards (glycidyl laurate and linolenate). In this

method LC-APCI-MS was used for detection of the

individual esters. Samples were cleaned over silica gel

columns. Even though results generally agreed well

there was no full correlation between any of the three

methods (Figure 3).In a further study, the DFA method was compared

with the ‘‘SGS 3-in-1 method’’ as well as to the DGF

C-III 18 (09) method. Regarding the DFA and theSGS methods, the comparison of results for sevensamples of different types of oils (two avocado, onepalm oil, one sunflower oil, one rice husk oil and twoDAG-rich oils) showed good correlation for all sam-ples except for the rice husk oil where the glycidolcontent obtained with the indirect method exceeded thevalue resulting from the direct approach by approxi-mately 30%. The correlation to the DGF C-III 18 (09),which was in the meantime withdrawn, was lesssatisfying and levels seemed to be underestimatedwith this method (Granvogl and Schieberle 2011a).

Direct and indirect methods for MCPD esters

The first comparison of direct and indirect analyticalmethods for MCPD esters was presented by Haineset al. (2011). The DGF method (C-III-18 (09)) appliedto measure the total MCPD equivalents (withoutdifferentiating MCPD and glycidyl esters) wasreported to give results that were consistently higherthan with the LC-TOFMS method that measuredMCPD and glycidyl esters directly. The LC-TOFMSmethod included two labelled standards (one labelledglycidyl ester and one labelled 3-MCPD di-ester).Samples were injected together with the labelledstandards after dilution without any clean-up.2-MCPD esters were not included in the analysis.

The new DGF method (C-VI 18 (10)) containedimprovements, through the implementation of alabelled 3-MCPD di-ester (d5-3-MCPD-1,2-bis-palmi-toyl ester), the use of chloride-free salts during saltingout in part B, and performing the derivatisationprocedure at room temperature in an organic solvent.

Figure 3. (Colour online). Comparison of 10 palm oil samples analysed by two direct methods (developed by DFA (Granvogland Schieberle 2011b) Nestle Research (Dubois et al. 2011) and one indirect analytical method developed by SGS (Kuhlmann2011a). The amounts of glycidyl esters detected by the different methods are expressed as total amounts of glycidol present in theglycidyl esters (mg kg�1).


However, neither of the modifications was sufficientto explain the huge discrepancies between the levelsof MCPD and glycidyl esters measured directly andindirectly. The authors (Haines et al. 2011) highlightedthe problem of rapid sensitivity losses due to fatresidues and the presence of sodium in the mobilephase. It is worth mentioning that the direct measure-ment approach did not consider the presence of2-MCPD esters, which have the same molecularweights as their analogous 3-MCPD esters. 2-MCPDesters are likely to co-elute with the 3-MCPD estersduring column chromatography, however, their signalresponse may be different (Dubois 2011).

Comparison data from a follow-up study werepresented at the 2011 OVID/BLL conference in Berlin/Germany (Hinrichsen 2011). This time, the new DGFmethod (C-VI 18 (10)) had been applied to measureMCPD and glycidyl esters indirectly. Comparison ofresults for ester-bound 3-MCPD from this new studyto those of the LC-TOFMS method mentioned aboveshowed that positive results in palm oil samples thathad been specially treated to remove 3-MCPD andglycidyl esters were obtained only by indirect analysis,and the direct analysis could not confirm their presenceabove a limit of 0.1mg kg�1. Both direct and indirectapproaches found glycidyl esters only at very low levelsif at all.

Recently, the development of a direct analyticalmethod for MCPD esters using a ‘‘dilute and shoot’’approach combined with LC-MS/MS for measurementusing standard additions with labelled and non-labelled standards was put forward (Pinkston andStoffolano 2011). The method, which did not consider2-MCPD esters, was compared with a modified versionof the original ‘‘Weisshaar method’’ where sodiumchloride is substituted by sodium sulphate (comparablewith DGF C-VI 18 (10) B). Results of nine vegetableoil samples presented showed in general a good

correlation but seemed to be biased, and valuesobtained from direct measurement were below thoseobtained by the applied indirect analysis. These effectsmay be explained by the fact that for direct analysisonly a limited set of reference compounds was avail-able. Furthermore, some 3-MCPD di-esters weredifficult to quantify due to some matrix interferences,indicating the possible need for implementation of aclean-up step prior to LC-MS/MS analysis.

Dubois (2011) recently presented the developmentof two direct analytical methods, the first targeting thedirect analysis of intact MCPD mono-esters andthe second one targeting the direct analysis of MCPDdi-esters. MCPD mono- and di-esters were isolated viadouble SPE (C18 and silica) or silica gel columns,respectively, and analysed by LC-TOFMS. Standardaddition quantification was used, with labelled internalstandards. The method was compared with an indirectanalytical method for 3- and 2-MCPD esters, whichwas based on the acid-catalysed transesterificationof the MCPD esters (Divinova et al. 2004), Extrelut�

extraction, derivatisation with HFBI, and GC-MS.For comparison, 30 different oil samples (mainly palmoil based) were analysed. The presence of 2-MCPDesters in the oils had been taken into consideration.The correlation between results showed a slope of0.975 and constant bias of �0.12mg g�1 indicatingthat the two methods provided very similar results(Figure 4).


So far only a very limited number of data for thecomparison of direct and indirect analytical methodsfor MCPD and glycidyl esters are available. With theexception of the study from Dubois on MCPD esters(Dubois 2011), the indirect methods subjected to inter-method comparison have been mainly alkaline based,

Figure 4. Determination of MCPD esters in 30 oil samples by the indirect method (acid-catalysed transesterification, HFBIderivatisation) and the direct method of Dubois et al. (2011). Regression calculations do not include coconut and palm kerneloil as these oils could not be quantified reliably by the present direct method (lack of specific analytical standards).

18 C. Crews et al.

which have been frequently modified and improved.With the exception of the study by Dubois, the directmethods used for comparison have not take intoconsideration the presence of 2-MCPD esters. Sincethese compounds carry the risk of co-elution with3-MCPD esters on liquid chromatography, andhave different signal intensities, a misinterpretation ofresults from these studies cannot be excluded.

Since direct analytical methods for glycidyl estershave shown to be relatively easy to handle andreference material more easily available, the compar-ison of direct and indirect analytical approaches mayin the future become more relevant for the MCPDester analysis. The indirect analysis of MCPD estersmay be impaired by the presence of glycidyl esters andby unidentified co-occurring compounds. The prepa-ration of adequate reference materials may thereforebecome difficult. Once indirect methods for MCPDesters that meet certain performance criteria have beenidentified and harmonised, their trueness could beassessed on naturally contaminated samples, and themethods compared with appropriate direct procedures.

Discussion

For glycidyl esters, in comparison to the directanalytical methods for MCPD esters direct analyticalmethods for glycidyl esters have been shown to requirea minimum of time and effort and a reasonably smallnumber of analytical standards (Dubois et al. 2011;Granvogl and Schieberle 2011b). Since direct analysisis not considered to be impaired by errors originatingfrom transesterification or derivatisation procedures itis favoured over indirect approaches for the quantifi-cation of glycidyl esters.

For MCPD esters, correlation without any signif-icant bias can so far be demonstrated only forcomparison of a direct LC-TOFMS method (samplepreparation over C18 and/or silica material) alsotaking into consideration the presence of 2-MCPDesters, versus an indirect method for 3 - and 2-MCPD

esters based on the cleavage of the MCPD esters underacidic conditions (Figure 5).

Indirect analytical methods require significantlyfewer chemical standards, to be less cumbersome, beeasily applicable to all type of different vegetable oilsand to allow easy distinction between 3- and 2- ‘‘esterbound MCPD.’’ Data showing a similarly goodcorrelation between other direct methods and alkalinebased transesterification methods such as the newDGF method (DGF C-VI 18 (10) Part B) or the ‘‘SGS3-in-1 method’’ are currently not available. However, acollection of data for different type of vegetable oils (26samples) showed an excellent correlation between the2 - and 3-MCPD esters determined by the ‘‘SGS 3-in-1method’’ and the acidic based transesterificationmethod mentioned above (Figure 5).

Analytical methods and occurrence data

Introduction

Early findings showed that refined palm oil is likely tobe the major dietary source of both MCPD esters andglycidyl esters. Samples of refined oils measured todate have shown high levels of the contaminants andthe oil is popular with food manufacturers. It is acommon ingredient of infant formulae and infantfollow-on formulae, which are often the major foodsource for this vulnerable population. Variable rangesof 3-MCPD esters have been found in certain foodgroups. They are not normally found in unrefinedvegetable oils or infant and baby foods in cans andjars. Higher but still relatively low levels have beenreported for infant formula, oil-based creamers, andheated cereals such as malt and bread crust. Thehighest levels have been found in refined vegetable oils,and particularly in palm oil.

Refined palm oils contain about 1–10mgkg�1 of3-MCPD esters with fractionated palm oils having less,e.g. palm oleins contained 0.4–0.6mg kg�1. A singlesurvey of 2-MCPD in 20 refined palm oils showed a

Figure 5. Comparison of values obtained from 26 refined vegetable oil samples analysed by the SGS 3-in-1 method and anacid-catalysed transesterification method (Divinova et al. 2004; Robert et al. 2004).


range of about 0.2–6mgkg�1. Glycidyl esters in palmoils range from 0.3 to 10mg kg�1 with up to15mgkg�1 in palm olein. Other refined oils generallycontain lower levels (typically 1–2mgkg�1) but withexceptional samples having about 20mgkg�1. Otherfoodstuffs analysed for MCPD and glycidyl esters arethose associated with high temperature processing or ahistory of free MCPD occurrence. Moderately highlevels of 3-MCPD esters have been reported in cookedpotato products (0.02–1.2mgkg�1), infant formulae(0.1–0.6mg kg�1), and toasted cereals (0.04–1.4mgkg�1). Only low levels have been reported inunrefined oil (50.3mg kg�1), most breads (50.011–0.042mgkg�1), and infant and baby food in cans andjars (50.01mg kg�1).

Tables 4–6 collate occurrence data published in2004–2011. Different analytical methods were usedfor the generation of these data and, thus care isrequired when considering the diversity and reliabilityof the data.

Analytical methods used for the occurrence data

The methods used for the first isolation and identifi-cation of 3-MCPD esters were based on TLC followedby GC-MS. They were applied to study the composi-tion of acid-HVP and to characterise the products ofthe reactions of fats and fat components with hydro-chloric acid in an investigation of the chlorinatedpropanols found in acid-HVP.

Following a revival in interest in the esters,methods based on cleavage of the acid/chloropropanolbond were devised and later methods enabling thedirect determination of individual esters have beenused on a small scale.

The TLC based method was not intended toprovide more than an indication of levels of MCPDesters that might be found in foods where free MCPDhad been encountered. They were carried out beforecharacterised standards and internal standards hadbeen prepared and without regard to the methodperformance and, thus quantification may be regardedas providing approximate data. The results obtainedshowed a very wide range of MCPD ester levels in thesame food type, and high levels (up to 6.4mgkg�1) inmeats that have not been evident in later analyses.

The first acid methanolysis based on indirectdetermination of MCPD esters without prior TLCisolation of fractions used in the analysis of foodstuffswas reported by Svejkovska et al. (2004). Both mono-and di-esters of 3-MCPD were found at levels of up toabout 35mgkg�1 of the fat portion (in French friesand dark malts). The detection limit was about1mgkg�1 of fat.

At the same time, enzyme cleavage was appliedby Hamlet and Sadd (2004) on the analysis of baked

cereal foods giving results comparable with theacid-catalysed transesterification methods used shortlyafter.

The alkaline transesterification causes rapiddecomposition of 3-MCPD esters, and early resultsusing this approach are probably unreliable (Hrncirıket al. 2011; Kuhlmann 2011a). Reduction of thetemperature improves the recovery after alkalinetransesterification considerably (Kuhlmann 2011a).

In the DGF method CIII 18b (2009), glycidyl esterswere determined indirectly in oils from measurementsof the difference between quantification of the sumof 3-MCPD and glycidyl esters, and determination of3-MCPD esters alone by avoiding sodium chlorideaddition, or by decomposition of glycidyl esters priorto transesterification.

Very many factors do or might have an effect onthe level of MCPD esters and glycidyl esters reported.These include:

. Fat extraction procedure.

. Methanolysis method – acid or basic orenzymatic.

. Methanolysis procedure reagent composition/concentration, reaction time and temperature.

. Addition of salt, or the choice of salt (foralkaline methanolysis).

. pH of the salting out solution (for alkalinemethanolysis).

. Method used to isolate and determine theMCPD produced.

However, it has been shown that high levels ofchloride can be used in acid methanolysis withoutoverestimation of 3-MCPD esters, and that glycidylesters do not interfere with the determination(Ermacora and Hrncirık 2012a). It has been shownthat the derivatisation procedure used for the determi-nation of the released free MCPD does not affect theresult (Seefelder et al. 2008).

Occurrence of 3-MCPD esters

Data on the occurrence of 3-MCPD esters in differentfoodstuffs are listed in Table 4. Data obtained with the2008 version of DGF method C-III-18 have not beenincluded in the table, because with this method, thesum of 3-MCPD and glycidol is determined. Althoughthousands of analyses of ester-linked 3-MCPD havebeen performed in recent years, only a small part of thedata have been published in peer reviewed journals orofficial reports. Data of 3-MCPD esters in thermallytreated foodstuffs other than fats and oils are sporadicand very heterogeneous due to differences in compo-sition and different thermal treatment of the singlefoodstuffs. Noticeable are high amounts found insalami and other microbiologically ripened foods;although the formation processes responsible are not

20 C. Crews et al.

Table

4.Occurrence

infood–ester-linked

3-M

CPD.

Class

Food

Method

Number

Range

(mgkg�1)

Comments

References

Cereals

Barley

Acidcleavage

80.044–1.386

Dolezalet

al.(2009)

Cereals

Biscuits

Acidcleavage

60.249–0.696

FSA

(2010)

Cereals

Breadassorted,UK

Acidcleavage

55

0.011–0.042

FSA

(2010)

Cereals

Breadtoasted

Enzymaticcleavage

70.06–0.16

Hamletet

al.(2004),HamletandSadd

(2004)

Cereals

Breakfast

cereals

Acidcleavage

50.04–0.888

FSA

(2010)

Cereals

Coffee

surrogates

Acidcleavage

50.145–1.184

Divinovaet

al.(2007)

Cereals

Crackers

TLC-G

C-M

S,acidcleavage

30.1–1.14

Reece

(2005),Svejkovskaet

al.(2004)

Cereals

Crispbread

TLC-G

C-M

S,acidcleavage

20.42–0.58

Reece

(2005),Svejkovskaet

al.(2004)

Cereals

Doughnuts

TLC-G

C-M

S,acidcleavage

20.42–1.21

Reece

(2005),Svejkovskaet

al.(2004)

Cereals

Rye,

wheat,flour

Acidcleavage/enzymatic

cleavage

34

50.005–1.02

Roasted

and

unroasted

Hamletet

al.(2004),HamletandSadd

(2004),Dolezalet

al.(2005,2009)

Coffee

Coffee

TLC-G

C-M

S,acidcleavage

26

50.1–0.39

Green,roasted

andsoluble

coffee

Reece

(2005),Svejkovskaet

al.(2004),

Dolezalet

al.(2005)

Dairy

Coffee

creamer

Acidcleavage

15

0.13–0.73

Karsulınovaet

al.(2007)

Dairy

Cream

(aerosol)

Acidcleavage

10

0.05–0.73

Karsulınovaet

al.(2007)

Dairy

Cheese

TLC-G

C-M

S,acidcleavage

5n.d.–1.28

Feta,Parm

esan,

processed

cheese

Reece

(2005),Svejkovskaet

al.(2004)

Infantandbabyfood

Cereal

Acidcleavage

55

0.011–0.23

FSA

(2010)

Infantandbabyfood

Infantbiscuits

Acidcleavage

20.11–0.306

FSA

(2010)

Infantandbabyfood

Jarred

food

Acidcleavage

55

0.011

FSA

(2010)

Infantandbabyfood

Humanbreast

milk

DirectGC-TOF-M

S126

50.011–0.076

Zelinkovaet

al.(2008)

Infantandbabyfood

Infantform

ula

Acidcleavage

11

50.075–0.588

Zelinkovaet

al.(2009a)

Infantandbabyfood

Milk,growing-up

Acidcleavage

30.062–0.291

Zelinkovaet

al.(2009a)

Infantandbabyfood

Infantform

ula

(fatportion)

DGF

C-III

18(09)

56

0.57–4.1

Fatcontent

23–28%

Weisshaar(2011),includingadditional

unpublished

data

Malt

Maltandbeer

Acidcleavage

20

0.004–0.65

Svejkovskaet

al.(2004),Divinovaet

al.

(2007),Dolezalet

al.(2009)

Meat

Chicken

grilled

TLC-G

C-M

S,acidcleavage

40.26–0.74

Reece

(2005),Svejkovskaet

al.(2004),

FSA

(2010)

Meat

Ham

TLC-G

C-M

S,acidcleavage

4n.d.–2.64

Smoked

and

unsm

oked

Reece

(2005),Svejkovskaet

al.(2004)

Meat

Salami

TLC-G

C-M

S,acidcleavage

60.88–6.41

Reece

(2005),Svejkovskaet

al.(2004),

Zelinkovaet

al.(2006)

Meat(fish)

Mackerel,herring

TLC-G

C-M

S,acidcleavage

30.28–1.08

Smoked

and

pickled

Reece

(2005),Svejkovskaet

al.(2004)

Miscellaneous

Bouilloncube

Acidcleavage

50.38–0.67

Karsulınovaet

al.(2007)

Miscellaneous

Nuts

roasted

Acidcleavage

4n.d.–0.5

Svejkovskaet

al.(2004),Zelinkovaet

al.

(2006)

Miscellaneous

Sweetspreads(fatportion)

DGF

C-III

18(09)

20

50.15–3.9

Fatcontent

30–45%


unpublished

data

Oilsandfats

Anim

alfats,unrefined

DGF

C-III

18(09)

25

50.1–0.14


unpublished

data

Oilsandfats

Cocoabutter

DirectLC-TOF

25

0.5

Haines

etal.(2011)

(continued

)


Table

4.Continued.

Class

Food

Method

Number

Range

(mgkg�1)

Comments

References

Oilsandfats

Fryingoils(fresh

andused)

DGF

C-III

18(09)

51

50.15–16.2


unpublished

data

Oilsandfats

Margarine(fatportion)

DGF

C-III

18(09)

22

50.15–7.7

Fatcontent

40–80%


unpublished

data

Oilsandfats

Mayonnaise(fatportion)

DGF

C-III

18(09)

17

50.15–1.04

Fatcontent

19–80%


unpublished

data

Oilsandfats

Palm

shortening/olein

DirectLC-TOF

50.4–0.6

Haines

etal.(2011)

Oilsandfats

Refined

coconutoil

Acidcleavage

21.418–1.694

Karsulınovaet

al.(2007)

Oilsandfats

Refined

palm

kernel

oil

Acidcleavage

30.85–1.40

Karsulınovaet

al.(2007)

Oilsandfats

Refined

palm

oil

Acidcleavage

41.39–4.17

Karsulınovaet

al.(2007)

Oilsandfats

Refined

vegetable

oils(notfrying)

DGF

C-III

18(09)

153

50.15–18.8


unpublished

data

Oilsandfats

Refined

vegetable

oils

Alkali/Br�

32

50.1–5.2

Kuhlm

ann(2011a)

Oilsandfats

Refined

hazelnut/walnutoil

Alkali/Br�

61.2–19.0

Kuhlm

ann(2011a)

Oilsandfats

Refined

oliveoil

Acidcleavage

55

0.3–2.462

Oliveoiland

olivepomace

oil

Zelinkovaet

al.(2006)

Oilsandfats

Refined

palm

oil

Alkali/Br�

20

1.1–10.0

Kuhlm

ann(2011a)

Oilsandfats

Refined

seed

oils

Acidcleavage

55

0.3–1.234

Zelinkovaet

al.(2006)

Oilsandfats

Refined

seed

oils

Alkali/Br�

15

50.1–2.1

Kuhlm

ann(2011a)

Oilsandfats

Refined

salm

onoil

Alkali/Br�

70.7–13.0

Kuhlm

ann(2011a)

Oilsandfats

Refined

vegetable

fats/oils

Acidcleavage

11

0.897–2.435

Differentiation

monoester–

di-ester

Seefelder

etal.(2008)

Oilsandfats

Unrefined

oils

Alkali/Br�

420

50.05

Kuhlm

ann(2011a)

Oilsandfats

Unrefined

vegetable

oils

DGF

C-III

18(09)

122

50.15–0.31


unpublished

data

Oilsandfats

Vegetable

oils

DirectLC-TOF

12

50.5

Haines

etal.(2011)

Oilsandfats

Virgin

oils

Acidcleavage

14

50.1

to5

0.3

Zelinkovaet

al.(2006)

Potato

products

Crisps

Acidcleavage

30

0.098–0.606

Dolezalet

al.(2008)

Potato

products

Crisps

Acidcleavage

20

0.048–1.186

FSA

(2010)

Potato

products

Crisps

Acidcleavage

16

0.229–1.008

Zelinkovaet

al.(2009b)

Potato

products

French

fries

Acidcleavage

20

0.035–0.397

FSA

(2010)

Potato

products

French

fries

Acidcleavage

16

0.1–0.258

Zelinkovaet

al.(2009b)

Potato

products

Homecooked

potato

products

Acidcleavage

17

0.014–0.225

FSA

(2010)

Potato

products

Mashed

Acidcleavage

30.038–0.275

Dolezalet

al.(2008)

Potato

products

Pre-fryingproducts

Acidcleavage

16

0.027–0.064

Zelinkovaet

al.(2009b)

22 C. Crews et al.

Table

5.Occurrence

infood–ester-linked

glycidol.

Class

Food

Method

Number

Range

(mgkg�1)

Comments

References

Infantandbabyfood

Infantform

ula

(fatportion)

DGF

C-III

18(09)

56

50.15–3.0

Fatcontent23–28%

Weisshaar(2011),includingaddi-

tionalunpublished

data

Miscellaneous

Sweetspreads(fatportion)

DGF

C-III

18(09)

20

50.15–2.1

Fatcontent30–45%


tionalunpublished

data

Oilsandfats

Anim

alfats,unrefined

DGF

C-III

18(09)

25

50.1


tionalunpublished

data

Oilsandfats

Cocoabutter

DirectLC-TOF

250.2

Haines

etal.(2011)

Oilsandfats

Cookingoil

DGF

C-III

18(09)

35–24

Shim

izuet

al.(2011)

Oilsandfats

Cookingoil

SPEþLC-M

S3

3–28

Shim

izuet

al.(2011)

Oilsandfats

Fryingoils(fresh

andused)

DGF

C-III

18(09)

51

50.15–10.7


tionalunpublished

data

Oilsandfats

Margarine(fatportion)

DGF

C-III

18(09)

22

50.15–5.0

Fatcontent40–80%


tionalunpublished

data

Oilsandfats

Mayonnaise(fatportion)

DGF

C-III

18(09)

17

50.15–0.33

Fatcontent19–80%


tionalunpublished

data

Oilsandfats

Palm

shortening/olein

DirectLC-TOF

60.4–15.6

Haines

etal.(2011)

Oilsandfats

Refined

vegetable

oils(not

frying)

DGF

C-III

18(09)

153

50.15–4.1


tionalunpublished

data

Oilsandfats

Refined

vegetable

oils

Alkali/Br�

32

50.1–3.1

Kuhlm

ann(2011a)

Oilsandfats

Refined

hazelnut/walnutoil

Alkali/Br�

60.5–1.4

Kuhlm

ann(2011a)

Oilsandfats

Refined

palm

oil

DGF

C-III

18(09)

36–8

Shim

izuet

al.(2011)

Oilsandfats

Refined

palm

oil

SPEþLC-M

S3

8–10

Shim

izuet

al.(2011)

Oilsandfats

Refined

palm

oil

Alkali/Br�

20

0.30–1.80

Kuhlm

ann(2011a)

Oilsandfats

Refined

seed

oils

Alkali/Br�

15

50.10–0.60

Kuhlm

ann(2011a)

Oilsandfats

Refined

salm

onoil

Alkali/Br�

750.10–1.20

Kuhlm

ann(2011a)

Oilsandfats

Unrefined

oils

Alkali/Br�

420

50.025

Kuhlm

ann(2011a)

Oilsandfats

Unrefined

vegetable

oils

DGF

C-III

18(09)

122

50.10


tionalunpublished

data

Oilsandfats

Vegetable

oils

DirectLC-TOF

10

50.2–3.7

Haines

etal.(2011)

Oilsandfats

Sunflower

oil

DirectLC-M

S4

1.2–2.1

GranvoglandSchieberle

(2011b)

Oilsandfats

Rapeseedoil

DirectLC-M

S3

0.2–0.36


(2011b)

Oilsandfats

Avocado

DirectLC-M

S4

2.5–7.8


(2011b)

Oilsandfats

Olive,

refined

DirectLC-M

S3

1.4–4.1


(2011b)


yet known. Data on oils, fats and foods containing

refined fats clearly show that the fat refining process is

the most important factor causing the formation of

3-MCPD esters. Refined palm oil and palm oil

fractions were identified as the most problematic

food ingredients for the dietary exposure to 3-MCPD

esters due to high contamination levels and the

widespread use of these fats.

Occurrence of glycidyl esters

Data on the occurrence of glycidyl esters are listed in

Table 5. Except for a handful of oil samples, all results

were obtained with indirect methods. The data clearly

show that the refining process is responsible not only

for the formation of 3-MCPD esters, but also for the

formation of glycidyl esters. Glycidyl esters have been

found only in refined vegetable oils and in products

containing them, including infant formulae, where

levels of up to about 3mgkg�1 have been reported.

Refined palm oils contained about 0.5–10mgkg�1 of

glycidyl esters with some identified frying oils contain-

ing almost 30mgkg�1. Other refined oils generally

contained up to 5mgkg�1.Data describing glycidyl ester levels in thermally

treated products except where derived from added

refined fats and oils have not been published so far.

Occurrence of 2-MCPD esters

Data on the occurrence of 2-MCPD esters, determined

in about 60 vegetable oils in a single study with an

indirect method (Kuhlmann 2011a), are listed in

Table 6. Ranges were between 0.2 and 6mgkg�1 for

refined palm oils and usually 50.5mgkg�1 for other

refined oils. Some other oil samples contained up to

11mgkg�1. Data on the occurrence of 2-MCPD esters

in thermally treated products excluding fats and oils

have not been published up to now.


It is evident that, up to now, only a small number of

reliable occurrence data have been made available in

peer reviewed journals or official reports. Current

knowledge of formation related to occurrence has been

summarised by Hamlet et al. (2011). Apart from theproblems of reliability and comparability of analytical

methods mentioned above, the most important gaps

are that there are only few and incidental occurrence

data on 3-MCPD esters in thermally treated foodstuffs

except refined fats and oils, and none on glycidyl esters

and 2-MCPD esters. The development of reliable

quantitative extraction procedures for different types

of food is a necessary precondition to closing this gap.Only few occurrence data of glycidyl esters are

available and only a handful of data of 2-MCPD esters

are available. As 2- and 3-MCPD can easily be

separated by GC-MS after derivatisation, it should

be possible to determine their esters simultaneously in

the same sample using indirect methods. Only very few

data have been published on 3-MCPD ester levels in

commercial food samples outside Europe.Analytical difficulties exist in that unless an official

method is available, because most investigators use

different approaches or even subtle changes that might

affect the result. The situation is compounded by the

paucity of well validated methods and the changes that

have been made to the official DGF (2009) over a short

period of time.

Discussion

The variety of factors that influence the quantitative

results of food sample analysis is so great that it is

difficult to analyse their effect for every survey

reported. Important factors are not restricted to the

analytical method. Conditions of edible oil refining

temperature and duration vary considerably, as can the

proportion of ingredient refined oils used in the foods,

Table 6. Occurrence in food – ester-linked 2-MCPD.

Class Food Method NumberRange

(mg kg�1) Comments Reference

Oils and fats Refined evening primrose oil Alkali/Br� 3 50.1–1.0 Kuhlmann (2011a)Oils and fats Refined grape kernel oil Alkali/Br� 4 0.4–2.4 Kuhlmann (2011a)Oils and fats Refined olive oil Alkali/Br� 4 50.1–0.4 Kuhlmann (2011a)Oils and fats Refined peanut oil Alkali/Br� 4 50.1–0.4 Kuhlmann (2011a)Oils and fats Refined palm oil Alkali/Br� 20 0.2–5.9 Kuhlmann (2011a)Oils and fats Refined rapeseed oil Alkali/Br� 45 50.1–0.3 Kuhlmann (2011a)Oils and fats Refined safflower oil Alkali/Br� 4 50.1–0.4 Kuhlmann (2011a)Oils and fats Refined soybean oil Alkali/Br� 45 50.1–0.1 Kuhlmann (2011a)Oils and fats Refined sunflower oil Alkali/Br� 45 50.1–0.3 Kuhlmann (2011a)Oils and fats Refined walnut/hazelnut oil Alkali/Br� 6 0.5–11 Kuhlmann (2011a)Oils and fats Refined salmon oil Alkali/Br� 7 0.1–0.3 Kuhlmann (2011a)

24 C. Crews et al.

and variations between different samples of the foodtype might be due just to this.

Analytical methods in biological samples

Background

Until very recently, analytical methods to analyseMCPD esters and glycidyl esters in biological samplesobtained from animal studies were simply not avail-able. Other than reports of 3-MCPD esters beingfound in fresh, unprocessed goat’s milk published inthe 1980s (Cerbulis et al. 1984), analytical methodsaddressed exclusively free 3-MCPD or glycidol andtheir metabolites after direct dosing of animals, andthose metabolites were determined mainly in urinesamples (see below). Only more recently, whenparticularly high levels of MCPD esters, and laterglycidyl esters, were found in refined vegetable oils thatpotentially contribute considerably to human foodexposure, has the fate of these compounds in theorganism received renewed attention. Their structuralsimilarity with lipids suggested the potential (partial)release of free 2-MCPD, 3-MCPD, or glycidol in thehuman gastrointestinal tract, but until very recently, itwas not known to what extent this release occurs, if itchanges the kinetics of their distribution, or if theesters themselves cause or contribute to specificadverse effects.

The metabolic fate and the potential toxicity of 3-MCPD and glycidyl esters as compared with their freecounterparts are being investigated in two majorstudies, a sub-chronic 90-day study (European FoodSafety Authority (EFSA) 2011) and a biokinetic studyperformed by the BfR. The fate and potential adverseeffects of 2-MCPD esters in animals or humans havenot at all been addressed to date.

Published studies involving analytical methodslinked to MCPD esters, glycidyl esters and theirmetabolites in biological samples (from animals orhumans, considering also non-processed milk as bio-logical sample) can be categorised as follows:

. In vivo studies addressing the metabolism offree 3-MCPD and glycidol.

. Occurrence of 3-MCPD esters in fresh, non-processed milk (goat, human).

. Analysis of 3-MCPD, glycidol, their esters,and their metabolites in tissues, blood andurines from animals treated with 3-MCPDand glycidyl esters.

. Analysis of biomarkers of exposure inhumans.

The following section briefly summarises thepathways involved in 3-MCPD and glycidol metabo-lism. Then those analytical methods for the parentcompounds and their metabolites with relevance to the

subject and issues today are discussed in more detailand summarised in Table 7.

Metabolic fate of 3-MCPD and glycidol

Most data on metabolism date back to the 1970s whenanalytical methods were far less developed than today,and more qualitative than quantitative. These studiesinvolved (1) following the fate of the radioactivity inanimals after administration of a radiolabelled parentcompound and characterisation/identification of theradioactivity in general or specific radiolabelled metab-olites in body fluids or tissues, or (2) analysis of theparent compound or a specific metabolite directly witha specific analytical method.

Early studies in the 1970s addressed the metabolicfate of 3-MCPD and the possible involvement ofglycidol as an obligatory reactive intermediate in itsdetoxification in rats and mice (Jones 1975; Jones et al.1978). The first urinary metabolites identified were thecysteine conjugates generated through glutathione(GSH) conjugation of glycidol as a possible interme-diate: these were the cysteine conjugate S-(2,3-dihy-droxypropyl)cysteine (DHPC) and the correspondingmercapturic acid N-acetyl-S-(2,3-dihydroxypropyl)cys-teine (DHPMA) that were identified in rat urine. It wasalso suggested that traces of glycerol were formed andexcreted in urine. A significant proportion of the 3-MCPD administered intraperitoneally was excretedunchanged in the urine, and significant quantities ofthe dose were exhaled as CO2 in rats and mice (Jones1975).

The conjugation to glutathione has been shown forseveral other mono- and dihalopropanols suggestingglycidol as a metabolic intermediate (Jones andFakhouri 1979). In addition, the only metabolicstudy available on 2-MCPD (Jones 1973) also identi-fied the mercapturic acid DHPMA as a urinarymetabolite, suggesting a common intermediate with3-MCPD, i.e. the epoxide glycidol. However, thetoxicities of 2 - and 3-MCPD are very different, andadditionally very different from the toxicity of glycidolby itself (reviewed in Schilter et al. 2011) suggestingthat glycidol is not relevant or insignificant as a (free)toxic intermediate. In parallel to the studies character-ising the metabolisation through GSH conjugation, anoxidative degradation pathway of 3-MCPD wasdiscovered trough transformation to the intermediates�-chlorolactaldehyde and �-chlorolactic acid resultingin excretion/exhalation of oxalic acid, CO2 and Cl�

(Jones et al. 1978).Metabolism studies on glycidol are limited, but

available data indicate that 3-MCPD and glycidolshare overlapping metabolic pathways. Glycidol isconverted to glycerol and exhaled as CO2 inrats and mice and produces the same GSH conjugates


Table

7.Analyticalapproaches

tostudyingthelevelsofMCPD,glycidol,theiresters

andmetabolitesin

biologicalsamples.

Analyte(s)

Internalstandard

Matrix/extraction

Transesterification

(tim

e)Derivatisation

Instrumentation

Comments

References

3-M

CPD

None

Humanurine;

3�ethyl

acetate

extraction

–NoneorBSTFA

GC-ECD

orGC-M

S(EI;SIM

)Calibrationusingspiked

humanblankurine

DeRooijet

al.

(1996)

3-M

CPD

3-M

CPD-d5

Raturine,

blood;silica

gel

column,ethylacetate

extraction

–HFBA

GC-M

S(N

CI;SIM

)Calibrationusingspiked

pooledblankratblood

orurine

Berger-Preiss

etal.(2010)

3-M

CPD,�-chloro-

lactic

acid,

DHPMA

3-M

CPD-d5,�-

chlorolactic

acid-d3,S-

DHPMA-d5

Raturine;

sample

dilution

1:100or1:1000

––

LC-M

S/M

S(N

IM;

SRM)

Additionally,anim

alswere

directlydosedwithdeu-

teratedcompounds

EFSA

(2011)

DHPMA

Benzylm

ercapturic

acid-d5

Humanurine;

acidification

topH

1.5–2with6N

HCl,lyophilisation,

neutralisationwith2N

NaOH,lyophilisation

anddesaltingin

methanol/acetone

methanolicHCl

(30min)

BSTFA

GC-M

S(EI;SIM

)Sensitivityinsufficientfor

humanurinesamples

from

workersexposedto

epichlorohydrin;inter-

feringsignals

DeRooijet

al.

(1997)

DHPMA

13C2-D

HPMA

Humanurine;

acidification

topH

2.5

withform

ate/

form

icacid,SPEwith

form

icacid/m

ethanol

elution

––

HIL

IC-ESI-MS/M

Sin

NIM

andMRM

Calibrationusingspiked

blankpooledhuman

urines

Eckertet

al.

(2010)

3-M

CPD

ester

3-M

CPD

Goatmilk;lyophilisation,

petroleum

ether

extrac-

tion,silica

gel

column

withhexane-benzene

1N

methanolicHCl

(16hRT)or

H2SO

4

–TLC

onsilica

gel

Gor

MS-D

CIGLC

for

FA

methylester

analysis

Cerbuliset

al.

(1984)

A)3-M

CPD;B)

3-M

CPD

ester

(determined

as

free

3-M

CPD

after

transesterifi-

cation);C)3-

MCPD

ester

determined

as

single

fattyacid

esterderivatives

3-M

CPD-d5;

PP-3-M

CPD,

2-dipalm

itate

Humanmilk;oxalate/etha-

nol/hexane-diethylether

extraction,solvent

exchangeto

hexane,

silica

gel

column,disso-

lutionin

THF

A)–;B)Sulphuric

acid/m

ethanol

(16h40�C);C)–

A)PBA;B)

PBA;C)–

GC/M

S(EI;SIM

)or

GCxGC

TOF-M

S(EI)

Determinationoffree

and

bound3-M

CPD

accord-

ingto

Zelinkovaet

al.

(2006)

Zelinkovaet

al.

(2008)

A)3-M

CPD;B)sum

ofglycidyland

3-M

CPD

esters

3-M

CPD-d5

A)cellculture

supernatants

dilutionwithsodium

phosphate

buffer;matrix

clean-upwithhexane-

acetone;

solvent

removal;B)cellculture

supernatants;lyophilisa-

tion,dissolutionin

t-butylm

ethylether/ethyl

acetate

A)–;B)NaOCH

3/

methanol

PBA

GC/M

S(EI;SIM

)A)accordingto

Divinova

etal.(2004);B)

Accordingto

DGF

MethodC-III-18(09)

Opt.A

Buhrkeet

al.

(2011)

N-(2,3-dihyroxypro-

pyl)valineof

globin

(2H5)N

-2,3-dihy-

droxypropyl-

adductsofglobin

(diH

OPrV

al)

Lysederythrocytes;globin

precipitation,ethylace-

tate

extraction,dissolu-

tionin

form

amide

–pentafluorophenyl

isothiocyanate,

diethylether

extraction,acety-

lationwithTEA

GC-M

S/M

S(N

CI)

N-terminalvalinebymodi-

fied

Edmandegradation.

Adduct

canbeform

edfrom

severalcompounds,

e.g.ECH,glycidol,

3-M

CPD,other

halohydrins

Landin

etal.

(1996)

as 3-MCPD, i.e. DHPC and DHPMA, in the urine ofrodents and humans (reviewed in Nomeir et al. 1995;Eckert et al. 2010). The metabolic pathways for 3-MCPD, 2-MCPD and glycidol are summarised inFigure 6 (adapted from Jones 1975; Jones et al. 1978;Lynch et al. 1998).

Some microorganisms are able to dehalogenate 3-MCPD to glycidol (van den Wijngaard et al. 1989),which was thought not to be a relevant reaction inhumans (Lynch et al. 1998). Recently, the possibility ofthe formation of a mercapturic acid conjugate from the�-chlorolactaldehyde intermediate has been raised(Habermeyer et al. 2011), but no experimental datasupporting this hypothesis was provided.

It is not entirely clear which metabolite(s) contrib-ute(s) to or cause the toxicities (renal, testicular) of 3-MCPD. While available data indicate the contributionof the oxidative pathway in the development ofreversible infertility in males of various animal speciesby inhibition of glycolysis in the epididymal tract(reviewed in Jones 1983), the causal involvement ofoxalic acid in the kidney carcinogenicity of 3-MCPD inrats was discussed but remains questionable.

The toxicity of glycidol has been studied since theearly 1970s (reviewed in Nomeir et al. 1995). Initialstudies on metabolism indicated the conversion ofglycidol to �-chlorohydrin (metabolite of 3-MCPD) by

stomach hydrochloric acid. This hypothesis was thor-oughly tested by Nomeir et al. (1995) demonstratingthat glycidol-derived radioactivity was mainly excretedin urine and exhaled as CO2. Urinary metaboliteprofiling identified one major and three lesser metab-olites (in total 15 metabolites were identified). Theindividual metabolites were not characterised further,but �-chlorolactic acid was quantitatively insignificantand independent of the route of administration indi-cating the absence of a significant conversion ofglycidol to 3-MCPD by hydrochloric acid in thestomach.

Metabolites of both the oxidative and the GSHconjugation pathway were recently confirmed in a90-day toxicological study with 3-MCPD and itsdipalmitate ester in rats (EFSA 2011). Free (but notbound) 3-MCPD, �-chlorolactic acid, and DHPMAwere analysed in urines at the end of the study.Interestingly, dosing of either free 3-MCPD or thedipalmitate ester resulted in similar profiles of thesemetabolites indicating that 3-MCPD is rapidly releasedfrom its ester and enters into the same metabolicpathways as 3-MCPD. The study also showed that theoxidative pathway is likely of minor importance, sinceonly low levels of �-chlorolactic acid close to the LOQwere found, while the mercapturic acid conjugateDHPMA was readily quantifiable at all dose levels of

Figure 6. Metabolic pathways for 3- and 2-MCPD and glycidol (adapted from Jones 1975; Jones et al. 1978; Lynch et al. 1998).GST, glutathione S-transferase; EH, epoxide hydrolase; NAT, N-acetyltransferase.


3-MCPD and its di-ester. The toxic effects of free andester-bound 3-MCPD were surprisingly similar andindicated no particular adverse effects specific to ester-bound 3-MCPD (palmitic di-ester). However, theseverity of the adverse effects was milder whendosing equimolar amounts of the di-ester indicatingthat the release kinetics may play a critical role in theirtoxicity. A current biokinetic study in rats conductedby the BfR is likely to confirm a relatively quickand complete release of the free compounds from both3-MCPD and glycidyl esters. These results seem tosubstantiate initial assumptions of the esters as addi-tional sources of dietary exposure to free MCPD andglycidol, respectively.

Analytical methods for 3-MCPD, glycidol,and their metabolites

The studies conducted in the 1970s were pivotal inidentifying the metabolic fate of 3-MCPD and glycidol.The methods used mostly involved TLC of urinarysamples (with or without solvent extraction) afteradministration of radioactively labelled compounds toexperimental animals. Identification of the analyteswas usually by comparison to the Rf values ofauthentic reference compounds, and quantificationwas by radioactivity scanning of the TLC plates orscintillation counting. Calcium oxalate was further-more identified by microscopic examination of diureticurine from treated animals (Jones 1975; Jones et al.1978; Nomeir et al. 1995). These methods are unlikelyto be of methodological relevance today; therefore, thefollowing sections focus on more modern state-of-the-art methods and include information on limits ofdetection and quantification that are likely applicabletoday.

3-MCPD, glycidol, and their metabolites in urineand blood

Non-radioactive, quantitative analysis of 3-MCPD (asa metabolite of epichlorohydrin) in rat urine was firstreported by De Rooij et al. (1996). Urine samples wererepeatedly extracted with ethyl acetate after addition ofinternal standard. Pooled organic phases were sub-jected directly to gas chromatography with electron-capture detection (GC-ECD). Alternatively, the ethylacetate extracts were silylated and subjected to GC-MSwith SIM detection. Calibration curves were obtainedfrom control urines spiked with authentic 3-MCPD.3-MCPD was identified by the presence of 5 charac-teristic ions in the mass spectrum of the trimethylsilylderivatives. The LOD of underivatised 3-MCPD was2 mgml�1 of urine, after TMS derivatisation the LODwas 0.4mgml�1 of urine and as low as 0.05mgml�1 ofurine when using higher injection volumes. Themethod was not sensitive enough to determine the

cysteine conjugates of 3-MCPD, DHPC, and DHPMA

in human urines (De Rooij et al. 1997). The methods

developed were proposed for use in occupational

settings for workers exposed to epichlorohydrin.Berger-Preiss et al. (2010) were recently able to

improve significantly the LOQ of 3-MCPD analysis in

urine and blood samples, which was achieved by GC-

MS with SIM in negative chemical ionisation mode

(GC-MS-NCI), with a reported LOQ of 2 ngml�1. The

method was designed for use in biokinetics studies in

rats. Urinary samples were acidified with acetic acid,

spiked with deuterated internal standard, absorbed on

to silica gel, and extracted with ethyl acetate. Analytes

were derivatised with heptafluorobutyric acid anhy-dride (HFBA). Calibration curves were established by

spiking blank rat blood or urine with different

concentrations of 3-MCPD and 3-MCPD-d5.

Precision, recovery, and accuracy were determined.

Blood samples were analysed in a similar way: water

and internal standard were added to blood mixed with

EDTA and samples were processed by addition of

silica gel as for urinary samples.In the recent 90-day toxicological study (EFSA

2011), urines were analysed directly for free 3-MCPD,

�-chlorolactic acid, and DHPMA by LC-MS/MS using

ionisation in negative ion mode and detection in

selected reaction mode (SRM). Samples were diluted1:100 or 1:1000, spiked with internal standards (3-

MCPD-d5 and S-DHPMA-d5), and injected. Limits of

detection were 1.73mg l�1 for 3-MCPD, 0.71 mg l�1 forS-DHPMA, and 9 mg l�1 for �-chlorolactic acid.

Eckert et al. (2010) recently reported the develop-

ment of a quantitative analytical method for the

mercapturic acid metabolite of glycidol (and 3-

MCPD) and DHPMA in human urine using hydro-

philic interaction liquid chromatography with tandem

mass spectrometry in negative electrospray ionisation

mode (HILIC-ESI-MS/MS). Urine samples were acid-

ified to a pH of 2.5 using ammonium formate buffer

and formic acid and internal standard (13C-DHPMA)

was added. Samples were processed by SPE. Afterelution, extracts were dried, reconstituted in solvent

and injected on to the LC-MS/MS. Two mass transi-

tions were used as quantifier and qualifier. Precision,

recovery, accuracy and the LOD for DHPMA

(5.5 mg l�1) were determined. The methods were

designed to study urinary metabolites of glycidol and

other alkylating chemicals in occupational settings and

in the general population (Eckert et al. 2011). Since

DHPMA is a common metabolite of 3-MCPD (and 2-

MCPD) and glycidol as well as their esters (and other

compounds, e.g. epichlorohydrin), it is not possible to

conclude which compounds humans were exposed to

without concurrent analysis of more specific bio-

markers of exposure or the levels of individual com-pounds in foods that were ingested.

28 C. Crews et al.

MCPD and glycidyl esters

To date only indirect methods have been available forthe determination of 3-MCPD esters in biologicalsamples.

3-MCPD esters in fresh milk (goat, human)

The first study to analyse 3-MCPD esters in a‘‘biological sample’’ dates back to 1984, whenCerbulis et al. (1984) described a class of compoundsseparated from the triacylglycerol fraction from freshnon-processed goat milk by TLC of the lipid fractionextracted by petroleum ether. It was not found inpeanut, corn, sunflower or safflower oils. They intro-duced the procedure of the release of free 3-MCPD bytransesterification overnight with methanolic hydro-chloric acid at room temperature followed by GC orTLC on silica gel G. 3-MCPD was identified by GC-MS. Alternatively, transesterification was carried outwith sulphuric acid as a catalyst. GC analyses of themethyl esters obtained from the transesterificationshowed that the major fatty acids present in 3-MCPDesters from goat’s milk were capric (C10:0), lauric(C12:0), palmitic (C14:0), stearic (C18:0), and oleicacid (C18:1), similar to the profile of goat milk TAG.The authors later identified 3-MCPD di-esters inhuman milk, but not in bovine milk or butter(Kuksis et al. 1986). As discussed above, this method(acid-catalysed transesterification) seems to be robustand specific, and the risk of interference from glycidylesters is considered low.

Zelinkova et al. (2008) analysed a series of humanbreast milk samples using GC-MS using SIM. Milk fatwas separated by extraction with potassium oxalate,ethanol and hexane-diethyl ether and re-extracted withethanol/hexane-diethyl ether. The extract was twicepartitioned with water, and the organic phase driedover anhydrous sodium sulphate, before dissolution inhexane. To analyse 3-MCPD di-esters, the milk fat wasspiked after extraction with 3-MCPD-d5-dipalmitate,applied to a silica gel column, washed with lightpetroleum ether, and eluted with diethyl ether. Thedried eluate containing 3-MCPD di-esters was re-dissolved in tetrahydrofuran (THF) and subjected toGC-MS. The method of Zelinkova et al. (2006) wasused to determine bound and free 3-MCPD by GC-MS. For free 3-MCPD the milk fat was extracted withhexane-acetone and derivatised with PBA for GC-MSdetermination. For bound 3-MCPD, the milk fat wasdissolved in THF, transesterified with sulphuric acid/methanol, neutralised with sodium bicarbonate, spikedwith 3-MCPD-d5 and derivatised with PBA. The LODwas determined to be 100 mg kg�1 milk fat with arelative standard deviation (RSD) of 5.9% and satis-factory linearity (r2¼ 0.9998). The calculated levels ofbound 3-MCPD in the milk samples ranged from 511to 76 mg kg�1. Di-esters prevailed over mono-esters,

and the fatty acids determined represented thoseusually found in human milk (lauric, palmitic, oleic).Based on these results, it is not possible to conclude towhich compounds individuals were exposed (esters orfree 3-MCPD). Concurrent analysis of free and boundMCPD and glycidol in the feed and food (andpotentially also in urine), respectively, would berequired. The fact that both in the goat and thehuman milk studies the fatty acid profile of bound 3-MCPD is similar to the endogenous milk fatty acidprofile may point to an endogenous (re-)synthesis ofthe fatty acid esters of 3-MCPD.

Free and bound 3-MCPD in cell culture supernatants

Parallel determination of free 3-MCPD and ester-bound 3-MCPD has been carried out in a recentin vitro study using the Caco-2 cell line as a model forhuman intestinal cells (Buhrke et al. 2011). In thisstudy, free 3-MCPD was determined from cell culturesupernatants by diluting the supernatant 10-fold with20% of sodium chloride in 20mM sodium phosphatebuffer at pH 6.0 and subsequent GC-MS analysisaccording to the protocol of Divinova et al. (2004)using 3-MCPD-d5 as internal standard. For theanalysis of ester-bound 3-MCPD, cell culture super-natants were lyophilised and resuspended in a mixtureof t-butylmethylether and ethyl acetate (80/20 v/v).This solution was used for ester cleavage and subse-quent 3-MCPD determination according to option Aof the later withdrawn DGF Standard Method CIII18b (DGF 2009) as described above. For analysis of 3-MCPD esters in biological samples a modification ofthe method would be desirable to comply with actualdevelopments.

Glycidyl esters

Comparable with the analysis of 3-MCPD esters,methods are available to determine glycidyl estersindirectly after ester cleavage and subsequent determi-nation of free glycidol. However, due to the highreactivity of the epoxide it is not simple to determineglycidol or glycidyl esters directly in biological sam-ples, because these substances can react with numerouscompounds present in biological systems. Therefore,indirect methods such as the determination of the urinemetabolite DHPMA (see above) or of haemoglobinadducts (see below) are employed for the estimation ofglycidol or glycidyl ester concentrations in biologicalsamples. Current improvements of indirect analyticalmethods minimise the conversion between glycidol and3-MCPD so that indirect methods should becomeapplicable in the near future for biological samples aswell. Otherwise, direct methods for the determinationof 3-MCPD and glycidyl esters omitting the step of


ester cleavage that are currently being developed couldbe applied to biological samples.

Biomarkers of exposure – haemoglobin adducts

As mentioned above, glycidol can hardly be directlydetected in biological systems due to its high reactivity.Glycidol belongs to the group of alkylating agents andcan therefore covalently bind to numerous biomole-cules such as proteins or DNA. As an example,glycidol can react with the N-terminal valine residueof haemoglobin. The resulting glycidol–haemoglobinadduct can be used as a biomarker to monitor theburden of glycidol in human blood. The generalprocedure to analyse haemoglobin adducts of alkylat-ing agents, such as ethylene oxide or 1,3-butadiene, hasbeen reviewed by Boogaard (2002). A specific methodfor the determination of haemoglobin adducts ofepichlorohydrin that are identical to the ones formedfrom glycidol has been published by the group ofLandin et al. (1996). Briefly, erythrocytes were isolatedfrom blood samples and subsequently the globinfraction was isolated from the erythrocytes. Afteraddition of the deuterated internal standard, a mod-ified Edman degradation procedure was carried out inthe presence of pentafluorophenyl isothiocyanate forderivatisation of N-(2,3-dihydroxypropyl)valine, whichis the product of glycidol covalently bound to theN-terminal valine residue of haemoglobin. The result-ing pentafluorophenylthiohydantoin is extracted andfurther modified to block the remaining hydroxylfunctions. Finally, the extract is analysed via GC-MSusing negative chemical ionisation (NCI), and theamount of the glycidol–haemoglobin adduct is calcu-lated from the derivatised Edman degradation productof the sample in relation to the corresponding Edmandegradation product of the deuterated internalstandard.

A recent study investigated the levels of bloodglycidol haemoglobin adducts (using the above men-tioned method by Landin et al. 1996) in humans whoconsumed DAG edible oils containing small amountsof glycidyl esters (Honda et al. 2011) over a period offour months. Background adducts were measurable inall, but no increased blood haemoglobin adducts weredetected in DAG oil consumers in comparison to non-consumers. On the contrary, background levels wereslightly higher in non-consumers. On the other hand, inthe study by Landin et al. (1996) smokers had higherlevels compared with non-smokers. However, the GElevels in DAG oils consumed were low, the sample sizewas small, and other dietary factors or potential othersources of glycidol haemoglobin adducts were notcontrolled.

As with many other biomarkers of exposure, thequantitative relationship between the biomarker and

the actual exposure is not straightforward.For example, erythrocytes are rather long-lived cells,therefore, the occurrence of haemoglobin adductsrepresents an average exposure over the full lifetimeof the cells, which is approximately 120 days.In addition, glycidol haemoglobin adducts are notspecific for the exposure to glycidol or its esters, butmight be formed through other compounds, such asepichlorohydrin. As long as the qualitative andquantitative relationship between the compound thatan organism is exposed to and a potential biomarker ofexposure is not known, its applicability has certainlylimitations.


Up to now, analytical methods to determine 3-MCPDand glycidyl esters have not been widely applied tosamples obtained from biological studies, and it is notyet established if the methods currently available foroils and foods are equally applicable to biologicalsamples, e.g. if the sample sizes obtained from animalstudies are quantitatively sufficient, or if the extractionmethods developed for oil or food matrices aretransferable to urine, blood, or tissues. The methodsemerging now as reliable and specific, be they direct orindirect, should be applied to biological samples to testtheir performance in these matrices.

Because MCPD, glycidol and their esters arepotentially quickly metabolised in the organism, itmay not be sufficient to determine the levels of theparent compounds in biological samples, but relevantmetabolites or biomarkers of exposure should beidentified. These metabolites or biomarkers shouldnot only reflect the actual exposure, but shouldoptimally also be connected to the putative adverseeffects relevant for human exposure through the diet.Mechanistic studies that are aimed at characterisingthe kinetics of the release of 3-MCPD or glycidol fromtheir esters must, therefore, be sufficiently specific.

Furthermore, the identification of analytical meth-ods for potential biomarkers in humans should alsotake into account that humans are likely exposed tomixtures of these compounds in foods. Refined oilshave been shown to contain esters of glycidol, 3-MCPD, and 2-MCPD at the same time. Many otherfood sources are more likely to contain the freecompounds, so the potential use of a metabolite thatis common to all of these compounds, such asDHPMA, as a biomarker of human exposure mightnot be adequate. The requirements for a relevanthuman biomarker are, however, far beyond the scopeof this paper and will not be discussed further.

The relevant ‘‘biological samples’’ (organs, bodyfluids) suitable to be analysed for specific purposes(mechanistic study, monitoring exposure, etc.) needs to

30 C. Crews et al.

be identified. Relevant metabolites and specific bio-markers of exposure need to be identified.

The appropriate analytical methods for thesemetabolites and biomarkers, to be applied to thebiological samples need to be developed and validated.Testing is needed of the applicability of direct andindirect methods for MCPD, glycidol and their estersin biological samples (sensitivity, specificity, etc.).Sample sizes (the quantity available from animalstudy or from humans) need to be determined, andextraction techniques for different tissues and bodyfluids to be designed to reduce matrix effects.

Consideration should be given to the study of theexposure to mixtures of MCPD, glycidol, their estersand resulting metabolites. Recent animal studies in ratsincluding that by EFSA (2011) will help to furtherrefine remaining questions and to narrow down andprioritise future research needs.

Conclusions

Research activity in this field over recent years hasbeen both intensive and productive. Comprehensivestudies of the reactions involved in the indirect analysisof MCPD esters have increased our understanding ofthe mechanisms involved, but many variations havebeen proposed and the need for harmonisation hasbecome apparent. Indirect methods for both boundMCPD and bound glycidol largely meet the require-ments for quick and routine analysis, and so indirectmethods are likely to be the ones used for routinescreening and food surveys. It can be consideredprudent that the wide scale application of suchmethods for this purpose has awaited better charac-terisation and validation. Indirect methods that pro-vide data on the content of both 2- and 3-MCPDesters, and hopefully glycidyl esters, are required andwill need validation by collaborative trial.

Direct methods for MCPD esters are likely to beused in a less routine fashion and the variability inperformance between different instruments and meth-ods means that a range of methods is likely to remain.These methods provide detailed information for for-mation and metabolism studies. The difficulties in thechromatographic separation of 2-MCPD esters from 3-MCPD esters in the direct methods remain to be dealtwith. For glycidyl esters the small number of esters andthe uncertainty of their analysis by the indirectmethods means that direct methods will continue tobe prominent. The growing commercial availability ofreference standards will benefit the scope and perfor-mance of the direct methods, but for both direct andindirect approaches there is a strong requirement forcharacterised reference materials and inter-laboratoryvalidation of one/two direct analytical method forglycidyl esters which include isotopically labelled

internal standards. The method(s) may be thoroughlycompared with indirect analytical methods such as the‘‘SGS 3-in-1 method,’’ which have already shown to becorrelated closely to the direct analytical methodsavailable. This may give support to laboratories whichare not equipped with highly sophisticatedinstruments.

The variation of results and doubts about methodperformance have to an extent been responsible for thelack of large-scale surveys of occurrence of MCPDesters and glycidyl esters. Much occurrence data havebeen derived from method testing experiments andstudies of formation. More data are required for onoils other than palm, and for composite foodstuffs and,where possible, this must be related to the processingconditions that affect their formation, in particularedible oil refining. It is very likely that the wide rangesof results reported truly represent a wide range ofconcentrations present in the foods analysed, and interms of using the data tabulated, the differences in theresults due to, for example, the presence or absence ofglycidyl esters is comparatively insignificant. Data of ahigher quality will undoubtedly be produced oncereliable analytical methods have been validated byinternational collaborative trials and applied to well-planned and -executed surveys.

Metabolic studies have shown that MCPD, glycidoland their esters are potentially quickly metabolised inthe organism. Both 3-MCPD and its dipalmitateexhibit nephrotoxicity and testicular toxicity, with theesters having milder effects than free 3-MCPD. Furtherstudies on the relevant metabolites and biomarkers ofexposure are required to calculate actual exposure andto characterise the kinetics of 3-MCPD and glycidolrelease from their esters. Monitoring of metabolitescommon to MCPD, glycidol and their esters might notgive sufficient information.

Very promising advances have been made towardsmitigation of MCPD ester and glycidyl ester formationin palm oil. Studies of all stages of palm oil productionhave indicated where contaminant formation can bereduced. It was considered that a multifacetedapproach might be beneficial, beginning with a reduc-tion of chloride application in fertiliser, continuingwith the use of plant varieties low in partial glycerideprecursors, the selection of young fruit of good quality,ending with technical changes to the refining steps, andthe removal by use of inorganic adsorbents.

The determination of 3-MCPD and glycidyl estersin biological studies using methods akin to those usedfor food is being investigated with caution. They havepromise but will need to be associated with methods todetermine the relevant biomarkers and metabolites.

It is over 30 years since the discovery of 3-MCPDand its esters in hydrolysed vegetable proteins, and theattention given to the free chloropropanol has in recentyears been superseded by interest in its esters in refined


vegetable oils. Dramatic reduction of the levels of freeMCPD in hydrolysed vegetable protein was achievedby close cooperation of the food industry withgovernment food safety agencies and research scien-tists, and the current repetition of this cooperation willhopefully lead to an effective mitigation of thisproblem.

Acknowledgements

This work was commissioned by the Process-relatedCompounds & Natural Toxins Task Force and RiskAssessment of Chemicals in Food Task Force of theEuropean branch of the International Life SciencesInstitute (ILSI Europe). Industry members of the taskforces are Ajinomoto Europe, Bunge Europe, Cargill,Danone, DSM, Kikkoman Foods Europe, Kraft FoodsEurope, Luigi Lavazza, Mars, Nestle, Pepsico International,Premier Foods, Procter & Gamble, Soremartec Italia –Ferrero Group, and Unilever. This review wascoordinated by Dr Alessandro Chiodini and Dr PratimaRao Jasti, Scientific Project Managers at ILSI Europe.For further information about ILSI Europe, pleaseemail: [email protected] or telephone þ32 2 771 00 14.

Note

The opinions expressed herein and the conclusions of thisreview are those of the authors and do not necessarilyrepresent the views of ILSI Europe or those of its membercompanies. Colin Crews, Michael Granvogl, Jan Kuhlmann,Alfonso Lampen and Rudiger Weisshaar received an hono-rarium from ILSI Europe for their participation in thisreview and reimbursement of their travel and accommoda-tion costs for attending the related meetings. AlessandroChiodini and Pratima Rao Jasti are employed by ILSIEurope.

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